Methods for enhancing exercise performance

ABSTRACT

Disclosed herein are methods for enhancing one or more effects of exercise in a subject by administering a PPARδ agonist (e.g., GW1516) to the subject in combination with an exercise program. Also disclosed are gene expression profiles unique to the combination of agonist-induced PPARδ activation and exercise. Such profiles are useful, at least, in methods for identifying the use of performance-enhancing drugs in exercised subjects (such as, professional or athletes). Direct interactions between PPARδ and exercised-induced kinases (e.g., AMPK or its subunits, AMPK α1 and/or AMPK α2) also are disclosed. Such protein-protein interactions provide new targets for identification of useful compounds.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.11/966,851, filed Dec. 27, 2007, now abandoned, which in turn claims thebenefit of U.S. Provisional Application No. 60/882,774 filed Dec. 29,2006, herein incorporated by reference.

ACKNOWLEDGEMENT OF GOVERNMENT SUPPORT

This work was supported by National Institutes of Health Grant No. 1 F32AR053803-01 (NRSA Fellowship). Therefore, the Government of the UnitedStates has certain rights in this invention.

REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAMLISTING APPENDIX SUBMITTED AS AN ASCII TEXT FILE

The Sequence Listing written in file 92150-876353_ST25.TXT, created onAug. 28, 2013, 71,772 bytes, machine format IBM-PC, MS-Windows operatingsystem, is hereby incorporated by reference in its entirety for allpurposes.

FIELD

This disclosure concerns the use of peroxisome proliferator-activatedreceptor (PPAR)δ agonists for improving exercise performance in asubject, methods for identifying substance-enhanced exercise performancein a subject, and methods for identifying compounds that affect theinteraction of PPARδ with exercise-induced kinases.

BACKGROUND

Skeletal muscle is an adaptive tissue composed of multiple myofibersthat differ in their metabolic and contractile properties includingoxidative slow-twitch (type I), mixed oxidative/glycolytic fast-twitch(type IIa) and glycolytic fast-twitch (type IIb) myofibers (Fluck etal., Rev. Physiol. Biochem. Pharmacol., 146:159-216, 2003; Pette andStaron, Microsc. Res. Tech., 50:500-509, 2000). Type I muscle fiberspreferentially express enzymes that oxidize fatty acids, contain slowisoforms of contractile proteins and are more resistant to fatigue thanare glycolytic muscle fibers (Fluck et al., Rev. Physiol. Biochem.Pharmacol., 146:159-216, 2003; Pette and Staron, Microsc. Res. Tech.,50:500-509, 2000). Type II fibers preferentially metabolize glucose andexpress the fast isoforms of contractile proteins (Fluck et al., Rev.Physiol. Biochem. Pharmacol., 146:159-216, 2003; Pette and Staron,Microsc. Res. Tech., 50:500-509, 2000).

Endurance exercise training triggers a complex remodeling program inskeletal muscle that progressively enhances performance in athletes suchas marathon runners, mountain climbers and cyclists. This involveschanges in metabolic programs and structural proteins within themyofibers that alter the energy substrate utilization and contractileproperties that act to reduce muscle fatigue (Fluck et al., Rev.Physiol. Biochem. Pharmacol., 146:159-216, 2003; Pette and Staron,Microsc. Res. Tech., 50:500-509, 2000). Training based adaptations inthe muscle are linked to increases in the expression of genes involvedin the slow-twitch contractile apparatus, mitochondrial respiration andfatty acid oxidation (Holloszy and Coyle, J. Appl. Physiol., 56:831-838,1984; Booth and Thomason, Physiol. Rev., 71:541-585, 1991; Schmitt etal., Physiol. Genomics, 15:148-157, 2003; Yoshioka et al., FASEB J.,17:1812-1819, 2003; Mahoney et al., FASEB J., 19:1498-1500, 2005;Mahoney and Tarnopolsky, Phys. Med. Rehabil. Clin. N. Am., 16:859-873,2005; Siu et al., J. Appl. Physiol., 97:277-285, 2004; Garnier et al.,FASEB J., 19:43-52, 2005; Short et al., J. Appl. Physiol., 99:95-102,2005; Timmons et al., FASEB J., 19:750-760, 2005). Such exercisetraining-related adaptations can improve performance and protect againstobesity and related metabolic disorders (Wang et al., PLoS Biol.,2:e294, 2004; Koves et al., J. Biol. Chem., 280:33588-33598, 2005).Moreover, skeletal muscles rich in oxidative slow-twitch fibers areresistant to muscle wasting (Minnaard et al., Muscle Nerve. 31: 339-48,2005).

PPARs are members of the nuclear receptor superfamily ofligand-inducible transcription factors. They form heterodimers withretinoid X receptors (RXRs) and bind to consensus DNA sites composed ofdirect repeats of hexameric DNA sequences separated by 1 bp. In theabsence of ligand, PPAR-RXR heterodimers recruit corepressors andassociated histone deacetylases and chromatin-modifying enzymes,silencing transcription by so-called active repression (Ordentlich etal., Curr. Top. Microbiol. Immunol., 254:101-116, 2001; Jepsen andRosenfeld, J. Cell Sci., 115:689-698, 2002; Privalsky, Ann. Rev.Physiol., 66:315-360, 2004). Ligand binding induces a conformationalchange in PPAR-RXR complexes, releasing repressors in exchange forcoactivators. Ligand-activated complexes recruit the basaltranscriptional machinery, resulting in enhanced gene expression. PPARsbind to lower-affinity ligands generated from dietary fat orintracellular metabolism. In keeping with their roles as lipid sensors,ligand-activated PPARs turn on feed-forward metabolic cascades toregulate lipid homeostasis via the transcription of genes involved inlipid metabolism, storage, and transport.

Three PPAR isotypes exist in mammals: α (also known as NR1C1), γ (alsoknown as NR1C3), and δ (also known as β or NR1C2). PPARδ is expressed inmost cell types with relative abundance (Smith, Biochem. Soc. Trans.,30(6):1086-1090, 2002), which led to early speculation that it may servea “general housekeeping role” (Kliewer et al., Proc. Natl. Acad. Sci.U.S.A., 91:7355-7359, 1994). More recently, PPARδ transgenic mousemodels and discoveries aided by the development of high-affinity PPARδagonists have revealed PPARδ as a key transcriptional regulator witheffects in diverse tissues including fat, skeletal muscle, and the heart(for review see, e.g., Barish et al., J. Clin. Invest., 116(3):590-597,2006).

Targeted expression of a constitutively active PPARδ receptor(VP16-PPARδ) transgene in rodent skeletal muscle promoted remodeling ofskeletal muscle to an oxidative phenotype and increased runningendurance in unexercised adult mice (Wang et al., PLoS Biol., 2:e294,2004). The observed PPARδ-mediated reprogramming of muscle fibersinvolved the increased expression of genes related to fatty acidoxidation, mitochondrial respiration, oxidative metabolism, andslow-twitch contractile apparatus (Wang et al., PLoS Biol., 2:e294,2004). These VP16-PPARδ transgenic mice, who had a phenotype similar toendurance-trained athletes, but who had had no exercise training,suggest that pharmacological activation of endogenous PPARδ in an adult,sedentary subject might provide an exercise effect without the actualexercise. Given the numerous benefits of exercise on general health,identification of orally active agents that mimic the effects ofexercise is a long standing, albeit elusive medical goal.

SUMMARY

This disclosure illustrates that, despite expectations to the contrary,pharmacological activation of endogenous PPARδ in adult, sedentarysubjects did not promote remodeling of skeletal muscle to an oxidativephenotype or increase running endurance in such subjects. Surprisingly,however, pharmacological activation of PPARδ in combination with atleast sub-maximal exercise synergistically modified skeletal musclearchitecture (e.g., induced fatigue resistant type I fiber specificationand mitochondrial biogenesis) and increased exercise performance (e.g.,running endurance). In addition, agonist-induced activation ofendogenous PPARδ in combination with exercise led to a unique “geneexpression signature” in skeletal muscle, which was distinct from thegene expression profile obtained by either exercise or drug intakealone, and revealed direct interactions between PPARδ andexercise-induced kinases (such as AMPK α1 and/or AMPK α2).

These and other discoveries described herein serve as the basis fordisclosed methods. For example, it can now be appreciated that PPARδagonists (e.g., GW1516) used in combination with exercise can enhanceexercise-induced effects, such as to improve exercise endurance (e.g.,running endurance) even more than may be achieved by exercise alone. Inanother example, the expression of one or more genes and/or proteinsthat are uniquely regulated by the combination of exercise and PPARδagonist administration can be used to identify subjects using drugs toenhance exercise performance. In still other examples, the newlyidentified protein complexes, including PPARδ and exercise-inducedkinases (such as AMPK α1 and/or AMPK α2), can be used to identify agentsthat have potential to affect PPARδ-regulated gene networks and thecorresponding downstream biochemical and/or physiological effects.

The foregoing and other features will become more apparent from thefollowing detailed description of several embodiments, which proceedswith reference to the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A is a series of bar graphs showing the effects of orallyadministered PPARδ agonist (GW1516) on mRNA expression levels of threebiomarkers of fatty acid oxidation, uncoupling protein 3 (UCP3),carnitine palmitoyl-transferase I (mCPT I), and pyruvate dehydrogenasekinase, isoenzyme 4 (PDK4), in quadriceps muscle isolated from sedentaryvehicle-treated (V), sedentary GW1516-treated (GW), sedentary VP16-PPARδtransgenic (TG), and sedentary wild-type littermates of VP16-PPARδtransgenic mice (WT). Data are presented as mean±SEM of N=4-9 mice eachanalyzed in triplicate. * Represents a statistically significantdifference between V and GW1516 groups (p<0.05, unpaired student'st-test), or TG and WT groups (p<0.05, unpaired student's t-test).

FIG. 1B-D are a series of bar graphs showing the regulation of oxidativegenes UCP3, mCPT I, and PDK4 by GW1516 (GW) in wild-type (WT) and PPARδnull (KO) primary muscle cells. * represents statistical significancebetween V and indicated groups (p<0.05, One Way ANOVA; post hoc:Dunnett's Multiple Comparison Test)

FIG. 1E is a series of bar graphs showing running endurance ofvehicle-treated sedentary (V; open bars) and GW1516-treated sedentary(GW; black bars) mice before (Week 0) and after (Week 5) treatment.Running endurance is quantified by the amount of time for which (leftpanel) or the distance (right panel) animals in each group ran on thetreadmill. Data is represented as mean±SD values from N=6 mice.

FIGS. 2A-C show the effects of administration of a PPARδ agonist,GW1516, on the gastrocnemius muscle of sedentary (V or GW) or trained(Tr or Tr+GW) mice. FIG. 2A shows digital images of representativemeta-chromatically stained frozen cross-sections of gastrocnemius musclefrom vehicle-treated, sedentary (V), GW1516-treated, sedentary (GW),vehicle-treated, exercised (Tr) and GW1516-treated, exercised (Tr+GW)mice. Type I (slow oxidative) fibers are darkly stained. FIG. 2B is abar graph showing the percentage of type I fibers (as a percentage ofthe total fibers) in V, GW, Tr, and Tr+GW gastrocnemius (N=3). FIG. 2Cis a bar graph showing the fold change in mitochondrial DNA to nuclearDNA ratio in V (left bar), GW (left center bar), Tr (right center bar),and Tr+GW (right bar) groups of mice (N=9). Data in (B) and (C) arepresented as mean±SEM. In each bar graph, * represents a statisticaldifference between V and the group(s) indicated by asterisk (p<0.05,One-Way ANOVA; post hoc: Dunnett's Multiple Comparison Test).

FIGS. 3A-C are a series of bar graphs showing gene expression inquadriceps muscle isolated from V, GW, Tr and Tr+GW groups. FIG. 3Ashows the relative gene expression levels of biomarkers for fatty acidoxidation (UCP3, mCPT I, PDK4; from left to right). FIG. 3B shows therelative gene expression levels of biomarkers for fatty acid storage(SCD1, FAS, SREBP1c). FIG. 3C shows the relative gene expression levelsof biomarkers for fatty acid uptake (FAT/CD36, LPL). Data is presentedas mean±SEM of N=9 mice, each analyzed in triplicate. * representsstatistically significant difference between V and the group(s)indicated by asterisk (p<0.05, One Way ANOVA; post hoc: Dunnett'sMultiple Comparison Test).

FIG. 3D shows images of Western blots illustrating protein expressionlevels of oxidative biomarkers (myoglobin, UCP3, CYCS, SCD1) and loadingcontrol (tubulin) in protein lysates prepared from quadriceps (N=3).

FIG. 4 shows a graph of muscle triglyceride levels in gastrocnemiusmuscle of V, GW, Tr and Tr+GW mice. Data is presented as mean±SEM of N=9mice, each analyzed in triplicate. * represents statistical significancebetween V and group(s) indicated by asterisk (*p<0.05, One Way ANOVA;post hoc:Dunnett's Multiple Comparison Test).

FIGS. 5A and B are bar graphs showing the effects of GW1516 treatment onrunning endurance in exercise-trained mice. Bar graphs of the (A) timeand (B) distance that vehicle-(V; open bars) and GW1516-treated (GW;black bars) mice ran on a treadmill before (Week 0) and after (Week 5)exercise training Data is represented as mean±SD of N=6 mice. ***represents statistically significant difference between V and GW groups(p<0.001; One Way ANOVA; post hoc:Tukey's Multiple Comparison Test).

FIG. 5C is a bar graph showing epididymal white adipose to body weightratio in V, GW, Tr and Tr+GW mice. Data is presented as mean±SEM of N=9mice, each analyzed in triplicate. * represents statistical significancebetween V and group(s) indicated by asterisk (*p<0.05, One Way ANOVA;post hoc:Dunnett's Multiple Comparison Test).

FIG. 5D shows digital images of H&E-stained cross-sections of epididymalwhite adipose from V, GW, Tr and Tr+GW mice. Similar results wereobtained from N=3 mice. * represents statistical significance between Vand group(s) indicated by asterisk (*p<0.05, One Way ANOVA; posthoc:Dunnett's Multiple Comparison Test).

FIG. 6 shows a Venn diagram comparing GW, Tr and Tr+GW target genesidentified in microarray analysis of quadriceps. Data is an average ofN=3 samples in each group. The selection criteria used a p<0.05 onBonferroni's multiple comparison test and a fold change greater than1.5.

FIG. 7A is a series of Western blot images showing AMPK activation byexercise. The levels of phospho-AMPK (phospho-AMPK) and total-AMPK inquadriceps muscle of sedentary (Sed/C57B1) and exercise-trained(Tr/C57B1) mice (N=5-7) are shown.

FIG. 7B is a series of Western blot images showing AMPK activation byVP16-PPARd over-expression. The levels of phospho-AMPK (phospho-AMPK)and total-AMPK in quadriceps muscle of sedentary wild-type or transgenicmice (Sed/WT or Sed/TG) are shown.

FIGS. 8A-B show the synergistic regulation of muscle gene expression byPPARδ and AMPK. (A) Venn diagram comparing GW, AI, and AI+GW targetgenes identified in microarray analysis of quadriceps. Data is anaverage of N=3 samples in each group. The selection criteria used ap<0.05 on Bonferroni's multiple comparison test and fold change greaterthan 1.5. (B) Comparison of Tr+GW and AI+GW dependent gene signaturesidentified in quadriceps. Data is an average of N=3 samples in eachgroup. The selection criteria used is similar to one used in FIG. 8A.

FIGS. 9A-H show the expression of (A) UCP3, (B) mCPT I, (C) PDK4, (D)SCD1, (E) ATP citrate lyase, (F) HSL, (G) mFABP, and (H) LPL transcriptsin quadriceps of mice treated with vehicle (V), GW1516 (GW), AICAR (AI)and the combination of the two drugs (GW+AI) for 6 days. Data ispresented as mean±SEM of N=6 mice in each group, analyzed intriplicate. * Indicates statistically significant difference between Vand indicated groups (p<0.05, One Way ANOVA; post hoc: Dunnett'sMultiple Comparison Test).

FIGS. 10A-L demonstrate the AMPK-PPARδ interaction. (A-D) show theexpression of metabolic genes in wild type and PPARδ null (KO) primarymuscle cells treated with V, GW, AI and GW+AI (bars from left to right)for 24 hours. In (E-F, J), AD293 cells were transfected withPPARδ+RXRα+Tk-PPRE along with control vector, AMPK α1, α2 and/or PGC1αas indicated above. (E) Induction of basal PPARδ transcriptionalactivity by AMPK α1 or α2. (F) Dose-dependent induction of PPARδtranscriptional activity is enhanced by AMPKα1 (closed circle) or AMPKα2 (closed square) compared to control (open triangle). In (G-I, K),AD293 cells were transfected and processed as indicated. (G-H)Representative blot showing co-immunoprecipitation of transfected (G) orendogenous (H) AMPK with Flag-PPARδ. (I) Metabolic p32 labeling of PPARδin AD293 cells transfected as described. (J) Synergistic regulation ofbasal (V) and ligand (GW) dependent PPARδ transcriptional activity byAMPK α2 subunit and PGC1α. (K) Co-immunoprecipitation of PPARδ but notAMPK α2 subunit with Flag-PGC1α. (L) Model depicting exercise-PPARδinteraction in re-programming muscle genome.

SEQUENCE INFORMATION

Nucleic acid and amino acid sequences may be referred to herein byGenBank accession number. It is understood that the sequences given suchGenBank accession numbers are incorporated by reference as they existedand were known as of Dec. 29, 2006.

DETAILED DESCRIPTION I. Introduction

Disclosed herein are methods for enhancing an exercise effect in asubject including the steps of performing by a subject physical activity(such as aerobic exercise (e.g., running)) sufficient to produce anexercise effect; and administering to the subject an effective amount ofa PPARδ agonist (e.g., GW1516). The exercise effect that is enhanced canbe, for example, improved running endurance (such as, improved runningdistance or improved running time or a combination thereof, increasedfatty acid oxidation in at least one skeletal muscle of the subject,and/or body fat (e.g., white adipose tissue) reduction). In some methodembodiments, a subject is a mammal (such as a racing mammal, like ahorse, a dog, or a human), and/or an adult, and/or an exercise-trainedsubject. In other exemplary methods, the PPARδ agonist is administeredon the same day(s) on which the physical activity is performed. In somemethods, administration of the PPARδ agonist is by oral administration,intravenous injection, intramuscular injection, and/or subcutaneousinjection. In other method embodiments, the effective amount of thePPARδ agonist is from about 1 mg per day to about 20 mg per day in asingle dose or in divided doses.

Also disclosed herein are methods for identifying the use ofperformance-enhancing substances in an exercise-trained subject, whichinclude determining in a biological sample taken from anexercise-trained subject (e.g, a skeletal muscle biopsy) the expressionof the molecules listed in Table 2 or listed in Table 4, or a subsetthereof, such as expression of at least 1, at least 5, at least 10, atleast 20, at least 40 of the molecules listed in Table 2 or in Table 4.

In some methods for identifying the use of performance-enhancingsubstances in an exercise-trained subject, (i) expression is upregulatedin one or more of (such as at least 5, at least 10, at least 20, atleast 35, or all of) adipose differentiation related protein;stearoyl-Coenzyme A desaturase 2; acetyl-Coenzyme A acetyltransferase 2;ATP citrate lyase; adiponectin, C1Q and collagen domain containing;diacylglycerol O-acyltransferase 2; lipase, hormone sensitive;monoglyceride lipase; resistin; CD36 antigen; fatty acid binding protein4, adipocyte; lipoprotein lipase; microsomal glutathione S-transferase1; GPI-anchored membrane protein 1; dual specificity phosphatase 7;homeodomain interacting protein kinase 3; insulin-like growth factorbinding protein 5; protein phosphatase 2 (formerly 2A), regulatorysubunit A (PR 65), beta isoform; protein tyrosine phosphatase-like(proline instead of catalytic arginine); member b; CCAAT/enhancerbinding protein (C/EBP), alpha; nuclear receptor subfamily 1, group D,member 2(Reverb-b); transferring; archain 1; solute carrier family 1(neutral amino acid transporter), member 5; RIKEN cDNA 1810073N04 gene;haptoglobin; retinol binding protein 4, plasma; phosphoenolpyruvatecarboxykinase 1, cytosolic; cell death-inducing DFFA-like effector c;interferon, alpha-inducible protein 27; carbonic anhydrase 3; cysteinedioxygenase 1, cytosolic; DNA segment, Chr 4, Wayne State University 53,expressed; dynein cytoplasmic 1 intermediate chain 2; Kruppel-likefactor 3 (basic); thyroid hormone responsive SPOT14 homolog (Rattus);cytochrome P450, family 2, subfamily e, polypeptide 1; complement factorD (adipsin); and/or transketolase; and/or (ii) expression isdownregulated in one or more of gamma-glutamyl carboxylase; 3-oxoacidCoA transferase 1; solute carrier family 38, member 4; annexin A7; CD55antigen; RIKEN cDNA 1190002H23 gene; fusion, derived from t(12; 16)malignant liposarcoma (human); lysosomal membrane glycoprotein 2; and/orneighbor of Punc E11, such as 1, 2, 3, 4, 5, 6, 7, 8 or 9 of thesemolecules.

Exemplary methods for identifying the use of performance-enhancingsubstances in an exercise-trained subject involve determining proteinexpression and/or determining expression of a gene encoding the protein.Such methods are routine in the art. In some examples, the level ofprotein or nucleic acid expression is quantified.

Methods of identifying an agent having potential to enhance exerciseperformance in a subject also are disclosed herein. Such methods caninclude (i) providing a first component comprising a PPARδ receptor oran AMPK-binding fragment thereof; (ii) providing a second componentcomprising an AMP-activated protein kinase (AMPK), AMPKα1, AMPKα2, or aPPARδ-binding fragment of any thereof; (iii) contacting the firstcomponent and the second component with at least one test agent underconditions that would permit the first component and the secondcomponent to specifically bind to each other in the absence of the atleast one test agent; and (iv) determining whether the at least one testagent affects the specific binding of the first component and the secondcomponent to each other. An effect on specific binding of the firstcomponent and the second component to each other identifies the at leastone test agent as an agent having potential to enhance exerciseperformance in a subject.

In some methods of identifying an agent having potential to enhanceexercise performance a third component, i.e., a PPARδ agonist (e.g.,GW1516), is involved and the first component, second component, andthird component are contacted as described above.

II. Abbreviations and Terms

AMPK AMP-activated protein kinase

bps Beats per second

MAPK Mitogen-activated protein kinase

mCPT I Muscle carnitine palmitoyl transferase I

QPCR or qPCR Quantitative PCR

PDK4 Pyruvate dehydrogenase kinase 4

PES Performance-enhancing substance(s)

PPAR Peroxisome proliferator-activated receptors

UCP3 Uncoupling protein 3

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which the disclosed subject matter belongs.Definitions of common terms in molecular biology may be found inBenjamin Lewin, Genes V, published by Oxford University Press, 1994(ISBN 0-19-854287-9); Kendrew et al. (eds.), The Encyclopedia ofMolecular Biology, published by Blackwell Science Ltd., 1994 (ISBN0-632-02182-9); and/or Robert A. Meyers (ed.), Molecular Biology andBiotechnology: A Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8). In order to facilitatereview of various embodiments of the disclosure, the followingexplanations of specific terms are provided:

Expression: The process by which the coded information of a nucleic acidtranscriptional unit (including, for example, genomic DNA or cDNA) isconverted into an operational, non-operational, or structural part of acell, often including the synthesis of a polypeptide. Gene expressioncan be influenced by external signals; for instance, exposure of a cell,tissue or subject to an agent that enhances gene expression. Expressionof a gene also may be regulated anywhere in the pathway from DNA to RNAto polypeptide. Regulation of gene expression occurs, for instance,through controls acting on transcription, translation, RNA transport andprocessing, degradation of intermediary molecules such as mRNA, orthrough activation, inactivation, compartmentalization or degradation ofspecific protein molecules after they have been made, or by combinationsthereof. Gene expression (for example expression of one or more of thegenes listed in Tables 2 and 4) can be measured at the RNA level or theprotein level and by any method known in the art, including, withoutlimitation, Northern blot, RT-PCR, Western blot, or in vitro, in situ,or in vivo protein activity assay(s).

The expression of a nucleic acid may be modulated compared to a controlstate, such as at a control time (for example, prior to administrationof a substance or agent that affects regulation of the nucleic acidunder observation) or in a control cell or subject, or as compared toanother nucleic acid. Such modulation includes but is not necessarilylimited to overexpression, underexpression, or suppression ofexpression. In addition, it is understood that modulation of nucleicacid expression may be associated with, and in fact may result in, amodulation in the expression of an encoded polypeptide or even apolypeptide that is not encoded by that nucleic acid (such as downstreamregulated polypeptide(s)).

The expression of a polypeptide also may be modulated compared to acontrol state, such as at a control time (for example, prior toadministration of a substance or agent that affects expression of anucleic acid encoding or regulating the polypeptide) or in a controlcell or subject, or as compared to another polypeptide. Modulation ofpolypeptide expression includes, but is not limited to, overexpressionor decreased expression of the polypeptide, alteration of thesubcellular localization or targeting of the polypeptide, alteration ofthe temporally regulated expression of the polypeptide (such that thepolypeptide is expressed when it normally would not be, or alternativelyis not expressed when it normally would be), alteration in the stabilityof the polypeptide, alteration in the spatial localization of theprotein (such that the polypeptide is not expressed where it wouldnormally be expressed or is expressed where it normally would not beexpressed).

Isolated: An “isolated” biological component (such as a polynucleotide,polypeptide, or cell) has been purified away from other biologicalcomponents in a mixed sample (such as a cell or tissue extract). Forexample, an “isolated” polypeptide or polynucleotide is a polypeptide orpolynucleotide that has been separated from the other components of acell in which the polypeptide or polynucleotide was present (such as anexpression host cell for a recombinant polypeptide or polynucleotide).

The term “purified” refers to the removal of one or more extraneouscomponents from a sample. For example, where recombinant polypeptidesare expressed in host cells, the polypeptides are purified by, forexample, the removal of host cell proteins thereby increasing thepercent of recombinant polypeptides in the sample. Similarly, where arecombinant polynucleotide is present in host cells, the polynucleotideis purified by, for example, the removal of host cell polynucleotidesthereby increasing the percent of recombinant polynucleotide in thesample. Isolated polypeptides or nucleic acid molecules, typically,comprise at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95% or even over 99% (w/w or w/v) of a sample.

Polypeptides and nucleic acid molecules are isolated by methods commonlyknown in the art and as described herein. Purity of polypeptides ornucleic acid molecules may be determined by a number of well-knownmethods, such as polyacrylamide gel electrophoresis for polypeptides, oragarose gel electrophoresis for nucleic acid molecules.

Sequence identity: The similarity between two nucleic acid sequences orbetween two amino acid sequences is expressed in terms of the level ofsequence identity shared between the sequences. Sequence identity istypically expressed in terms of percentage identity; the higher thepercentage, the more similar the two sequences.

Methods for aligning sequences for comparison are well known in the art.Various programs and alignment algorithms are described in: Smith andWaterman, Adv. Appl. Math. 2:482, 1981; Needleman and Wunsch, J. Mol.Biol. 48:443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci. USA85:2444, 1988; Higgins and Sharp, Gene 73:237-244, 1988; Higgins andSharp, CABIOS 5:151-153, 1989; Corpet et al., Nucleic Acids Research16:10881-10890, 1988; Huang, et al., Computer Applications in theBiosciences 8:155-165, 1992; Pearson et al., Methods in MolecularBiology 24:307-331, 1994; Tatiana et al., (1999), FEMS Microbiol. Lett.,174:247-250, 1999. Altschul et al. present a detailed consideration ofsequence alignment methods and homology calculations (J. Mol. Biol.215:403-410, 1990).

The National Center for Biotechnology Information (NCBI) Basic LocalAlignment Search Tool (BLAST™, Altschul et al., J. Mol. Biol.215:403-410, 1990) is available from several sources, including theNational Center for Biotechnology Information (NCBI, Bethesda, Md.) andon the Internet, for use in connection with the sequence-analysisprograms blastp, blastn, blastx, tblastn and tblastx. A description ofhow to determine sequence identity using this program is available onthe internet under the help section for BLAST™.

For comparisons of amino acid sequences of greater than about 30 aminoacids, the “Blast 2 sequences” function of the BLAST™ (Blastp) programis employed using the default BLOSUM62 matrix set to default parameters(cost to open a gap [default=5]; cost to extend a gap [default=2];penalty for a mismatch [default=−3]; reward for a match [default=1];expectation value (E) [default=10.0]; word size [default=3]; number ofone-line descriptions (V) [default=100]; number of alignments to show(B) [default=100]). When aligning short peptides (fewer than around 30amino acids), the alignment should be performed using the Blast 2sequences function, employing the PAM30 matrix set to default parameters(open gap 9, extension gap 1 penalties). Proteins with even greatersimilarity to the reference sequences will show increasing percentageidentities when assessed by this method.

For comparisons of nucleic acid sequences, the “Blast 2 sequences”function of the BLAST™ (Blastn) program is employed using the defaultBLOSUM62 matrix set to default parameters (cost to open a gap[default=11]; cost to extend a gap [default=1]; expectation value (E)[default=10.0]; word size [default=11]; number of one-line descriptions(V) [default=100]; number of alignments to show (B) [default=100]).Nucleic acid sequences with even greater similarity to the referencesequences will show increasing percentage identities when assessed bythis method.

Specific binding: Specific binding refers to the particular interactionbetween one binding partner (such as a binding agent) and anotherbinding partner (such as a target). Such interaction is mediated by oneor, typically, more noncovalent bonds between the binding partners (or,often, between a specific region or portion of each binding partner). Incontrast to non-specific binding sites, specific binding sites aresaturable. Accordingly, one exemplary way to characterize specificbinding is by a specific binding curve. A specific binding curve shows,for example, the amount of one binding partner (the first bindingpartner) bound to a fixed amount of the other binding partner as afunction of the first binding partner concentration. As the firstbinding partner concentration increases under these conditions, theamount of the first binding partner bound will saturate. In anothercontrast to non-specific binding sites, specific binding partnersinvolved in a direct association with each other (e.g., aprotein-protein interaction) can be competitively removed (or displaced)from such association (e.g., protein complex) by excess amounts ofeither specific binding partner. Such competition assays (ordisplacement assays) are very well known in the art.

The singular terms “a,” “an,” and “the” include plural referents unlesscontext clearly indicates otherwise. Similarly, the word “or” isintended to include “and” unless the context clearly indicatesotherwise. “Comprising” means “including.” Hence “comprising A or B”means “including A or B”, or “including A and B.”

Materials, methods, and examples are illustrative only and not intendedto be limiting. Methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of the presentdisclosure (see, e.g., Sambrook et al., Molecular Cloning: A LaboratoryManual, 2d ed., Cold Spring Harbor Laboratory Press, 1989; Sambrook etal., Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring HarborPress, 2001; Ausubel et al., Current Protocols in Molecular Biology,Greene Publishing Associates, 1992 (and Supplements to 2000); Ausubel etal., Short Protocols in Molecular Biology: A Compendium of Methods fromCurrent Protocols in Molecular Biology, 4th ed., Wiley & Sons, 1999;Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory Press, 1990; and Harlow and Lane, Using Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratory Press, 1999).

III. Methods of Enhancing an Exercise Effect

Exercise is known to have many effects on subjects that perform it.Exercise effects at the molecular, biochemical, and/or cellular levels(e.g., modified regulation of genes and/or gene networks andcorresponding proteins involved in energy substrate utilization andcontractile properties of muscle) form the basis of physiologicaleffects that are observed at the tissue, organ, and/or whole body levels(e.g., increased cardiorespiratory endurance, muscular strength,muscular endurance, and/or flexibility, and/or improvements in bodyappearance). Disclosed herein are methods for enhancing one or moreexercise effects by combining, at least, physical activity withadministration of one or more PPARδ agonists. In some examples, physicalactivity is replaced with administration of an AMPK activator (e.g.,AICAR).

In general terms, exercise is the performance of some physical activity.A single episode (also referred to as a bout) of physical activity isperformed for a particular duration and at a particular intensity. Ifmore than one bout of exercise is performed, separate bouts of exercisemay have the same or different durations and/or the same or differentintensities.

In some method embodiments, a single bout of exercise may last for up to30 minutes, up to 45 minutes, up to 60 minutes, up to 90 minutes, up to2 hours, up to 2.5 hours, up to 3 hours, or even longer. Typically, inthe absence of a prior exercise history, repeated episodes of physicalactivity are needed to achieve an exercise-induced effect (such as,increased aerobic capacity or increase running endurance). Thus, in somedisclosed methods, bouts of physical activity may be repeated within asingle day; for instance, up to 2 bouts of exercise per day, up to 3bouts of exercise per day, up to 4 bouts of exercise per day, up to 5bouts of exercise per day, or even more bouts per day. Some professionalathletes or racing mammals may exercise in repeated bouts for a total of8 hours or more a day. In other method embodiments, bouts (or repeatedbouts) of exercise are performed on a daily basis, 6 times per week, 5times per week, 4 times per week or 3 times per week. In at least someof the disclosed methods, exercise may continue for at least 2 weeks,for at least 4 weeks, for at least 6 weeks, for at least 3 months, forat least 6 months, for at least 1 year, for at least 3 years, orindefinitely (for the lifetime of the subject).

Exercise generally is performed at an intensity that is more than theusual (e.g., average, median, normal standard, or normoactive) activityfor a subject, and/or at or less than the maximum activity achievable bya subject performing a particular exercise. Any known indicator ofphysical performance can be used to determine whether a subject isperforming more than a usual amount of activity, including, forinstance, measuring heart rate, repetition rate (e.g., revolutions persecond, minutes per mile, lifts per minute, and many others), and/orforce output. In some methods, a bout of exercise is performed atsub-maximal intensity; for instance, at about 10% maximal intensity, 25%maximal intensity, 50% maximal intensity, or 75% maximal intensity. Inother methods, a bout of exercise is performed at 40%-50% maximal heartrate, 50%-60% maximal heart rate, 60%-70% maximal heart rate, or 75%-80%maximal heart rate, where maximum heart rate for a human subject iscalculated as: 220 bps—(age of the subject).

Exercise is generally grouped into three types: (i) flexibility exercise(such as, stretching), which is believed to, at least, improve the rangeof motion of muscles and joints; (ii) aerobic exercise; and (iii)anaerobic exercise (such as, weight training, functional training orsprinting) which is believed to, at least, increase muscle strength andmass.

Aerobic exercise refers to a physical activity in which oxidative oraerobic metabolism (as compared to glycolytic or anaerobic metabolism)substantially predominates in exercised skeletal muscles. In particularmethod embodiments, a subject performs one or more aerobic exercises.Exemplary aerobic exercises include, without limitation, aerobics,calisthenics, cycling, dancing, exercise machines (rowing machine,cycling machine (e.g., inclined or upright), climbing machine,elliptical trainers, and/or skiing machines), basketball, football,baseball, soccer, footbag, housework, jogging, martial arts, massage,pilates, rowing, running, skipping, swimming, walking, yoga, boxing,gymnastics, badminton, cricket, track and field, golf, ice hockey,lacrosse, rugby, tennis, or combinations thereof.

The disclosed methods contemplate enhancing any known or observableeffect of exercise (such as an aerobic exercise, like walking orrunning). In particular methods, running endurance (e.g., runningdistance and/or running time) is enhanced.

Enhancing an exercise effect (such as running endurance) means that sucheffect is improved in a subject more than would have occurred byexercise alone. In some method embodiments, an enhanced exercise effectis determined by discontinuing administration of a PPARδ agonist in thesubject and observing (e.g., qualitatively or quantitatively) areduction in the exercise effect of interest (e.g., aerobic endurance,such as running endurance). In some instances, an exercise effect ofinterest, the PPARδ-enhanced portion of which is lost upondiscontinuance of PPARδ administration, will be reduced by at leastabout 5%, by at least about 10%, by at least about 20%, by at leastabout 30%, or by at least about 50% as compared to the magnitude of theeffect with exercise alone.

A. PPARδ Agonists

The disclosed methods envision the use of any PPARδ agonist. Preferablysuch agonist would be non-toxic in the subject to which it isadministered. Exemplary PPARδ agonists include GW1516, L-165041 (asdescribed by, e.g., Leibowitz et al., FEBS Lett., 473(3):333-336, 2000),any one or more compounds described in PCT Publication Nos.WO/2006/018174, WO/2005/113506, WO/2005/105754, WO/2006/041197,WO/2006/032023, WO/01/00603, WO/02/092590, WO/97/28115, WO/97/28149,WO/97/27857, WO/97/28137, WO/97/27847, and/or WO/98/27974, and/or apublished U.S. national phase application or issued U.S. patentcorresponding to any of the foregoing (each of which is expresslyincorporated herein by reference). Moreover, other PPARδ agonists can beidentified using the methods described, for example, in PCT PublicationNo. WO/1998/049555 or any corresponding published U.S. national phaseapplication or issued U.S. patent (each of which is expresslyincorporated herein by reference).

In a specific example, the PPARδ agonist is GW1516 (also referred to inthe art as GW501516). GW1516 is(2-methyl-4(((4-methyl-2-(4-trifluoromethylphenyl)-1,3-thiazol-5-yl)methyl)sulfanyl)phenoxy)aceticacid as has been shown to be is bioactive in humans (Sprecher et al.,Arterioscler. Thromb. Vasc. Biol. 27(2): 359-65, 2007). In specificexamples, GW1516 is administered orally, for example 1 mg-20 mg/day,such as 2.5 mg or 10 mg per day.

B. Subjects

The disclosed methods can be performed in any subject capable ofperforming physical activity (e.g., aerobic exercise). In some methodembodiments, a subject is a living multi-cellular vertebrate organism(e.g., human and/or non-human animals). In other exemplary methods, asubject is a mammal (including humans and/or non-human mammals such asveterinary or laboratory mammals) or, in more particular examples, aracing mammal (such as a horse, a dog, or a human). In still othermethods, a subject is an adult, an exercise-trained subject, or ahealthy subject. Some representative adult, human subjects are 16 yearsold or old, 18 years old or older, or 21 years old or older. Somerepresentative exercised-trained subjects have performed physicalactivity (such described in detail above) for at least 4 weeks, for atleast 6 weeks, for at least 3 months, or for at least 6 months. In someexamples the subject is healthy, for example, is a subject in which noknown disease or disorder has been diagnosed or would be apparent afterreasonable inquiry to an ordinarily skilled physician in the field towhich the disease or disorder pertains.

C. Methods of Administration, Formulations and Dosage

The disclosed methods envision the use of any method of administration,dosage, and/or formulation of PPARδ agonist that has the desired outcomeof enhancing an exercise effect in a subject receiving the formulation,including, without limitation, methods of administration, dosages, andformulations well known to those of ordinary skill in the pharmaceuticalarts.

Modes of administering a PPARδ agonist (or a formulation including aPPARδ agonist) in a disclosed method include, but are not limited to,intrathecal, intradermal, intramuscular, intraperitoneal (ip),intravenous (iv), subcutaneous, intranasal, epidural, intradural,intracranial, intraventricular, and oral routes. In a specific examplethe PPARδ agonist is administered orally. Other convenient routes foradministration of a PPARδ agonist (or a formulation including a PPARδagonist) include for example, infusion or bolus injection, topical,absorption through epithelial or mucocutaneous linings (for example,oral mucosa, rectal and intestinal mucosa, and the like) ophthalmic,nasal, and transdermal. Administration can be systemic or local.Pulmonary administration also can be employed (for example, by aninhaler or nebulizer), for instance using a formulation containing anaerosolizing agent.

In specific method embodiments, it may be desirable to administer aPPARδ agonist locally. This may be achieved by, for example, local orregional infusion or perfusion, topical application (for example, wounddressing), injection, catheter, suppository, or implant (for example,implants formed from porous, non-porous, or gelatinous materials,including membranes, such as sialastic membranes or fibers), and thelike.

In other method embodiments, a pump (such as a transplanted minipump)may be used to deliver a PPARδ agonist (or a formulation including aPPARδ agonist) (see, e.g., Langer Science 249, 1527, 1990; Sefton Crit.Rev. Biomed. Eng. 14, 201, 1987; Buchwald et al., Surgery 88, 507, 1980;Saudek et al., N. Engl. J. Med. 321, 574, 1989). In another embodiment,a PPARδ agonist (or a formulation including a PPARδ agonist) isdelivered in a vesicle, in particular liposomes (see, e.g., Langer,Science 249, 1527, 1990; Treat et al., in Liposomes in the Therapy ofInfectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss,N.Y., pp. 353-365, 1989).

In yet another method embodiment, a PPARδ agonist can be delivered in acontrolled-release formulation. Controlled-release systems, such asthose discussed in the review by Langer (Science 249, 1527 1990), areknown. Similarly, polymeric materials useful in controlled-releasedformulations are known (see, e.g., Ranger et al., Macromol. Sci. Rev.Macromol. Chem. 23, 61, 1983; Levy et al., Science 228, 190, 1985;During et al., Ann. Neurol. 25, 351, 1989; Howard et al., J. Neurosurg.71, 105, 1989). For example, a PPARδ agonists may be coupled to a classof biodegradable polymers useful in achieving controlled release of acompound, including polylactic acid, polyglycolic acid, copolymers ofpolylactic and polyglycolic acid, polyepsilon caprolactone, polyhydroxybutyric acid, polyorthoesters, polyacetals, polydihydropyrans,polycyanoacrylates and cross-linked or amphipathic block copolymers ofhydrogels.

The disclosed methods contemplate the use of any dosage form of PPARδagonist (or formulation containing the same) that delivers the PPARδagonist and achieves a desired result. Dosage forms are commonly knownand are taught in a variety of textbooks, including for example, Allenet al., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems,Eighth Edition, Philadelphia, Pa.:Lippincott Williams & Wilkins, 2005,738 pages. Dosage forms for use in a disclosed method include, withoutlimitation, solid dosage forms and solid modified-release drug deliverysystems (e.g., powders and granules, capsules, and/or tablets);semi-solid dosage forms and transdermal systems (e.g., ointments,creams, and/or gels); transdermal drug delivery systems; pharmaceuticalinserts (e.g., suppositories and/or inserts); liquid dosage forms (e.g.,solutions and disperse systems); and/or sterile dosage forms anddelivery systems (e.g., parenterals, and/or biologics). Particularexemplary dosage forms include aerosol (including metered dose, powder,solution, and/or without propellants); beads; capsule (includingconventional, controlled delivery, controlled release, enteric coated,and/or sustained release); caplet; concentrate; cream; crystals; disc(including sustained release); drops; elixir; emulsion; foam; gel(including jelly and/or controlled release); globules; granules; gum;implant; inhalation; injection; insert (including extended release);liposomal; liquid (including controlled release); lotion; lozenge;metered dose (e.g., pump); mist; mouthwash; nebulization solution;ocular system; oil; ointment; ovules; powder (including packet,effervescent, powder for suspension, powder for suspension sustainedrelease, and/or powder for solution); pellet; paste; solution (includinglong acting and/or reconstituted); strip; suppository (includingsustained release); suspension (including lente, ultre lente,reconstituted); syrup (including sustained release); tablet (includingchewable, sublingual, sustained release, controlled release, delayedaction, delayed release, enteric coated, effervescent, film coated,rapid dissolving, slow release); transdermal system; tincture; and/orwafer.

Typically, a dosage form is a formulation of an effective amount (suchas a therapeutically effective amount) of at least one activepharmaceutical ingredient (such as a PPARδ agonist) withpharmaceutically acceptable excipients and/or other components (such asone or more other active ingredients). The preferred aim of a drugformulation is to provide proper administration of an active ingredient(such as a PPARδ agonist) to a subject. A formulation should suit themode of administration. The term “pharmaceutically acceptable” meansapproved by a regulatory agency of the federal or a state government orlisted in the U.S. Pharmacopoeia or other generally recognizedpharmacopoeia for use in animals, and, more particularly, in humans.

Excipients for use in exemplary formulations include, for instance, oneor more of the following: binders, fillers, disintegrants, lubricants,coatings, sweeteners, flavors, colorings, preservatives, diluents,adjuvants, and/or vehicles. In some instances, excipients collectivelymay constitute about 5%-95% of the total weight (and/or volume) of aparticular dosage form.

Pharmaceutical excipients can be, for instance, sterile liquids, such aswater and/or oils, including those of petroleum, animal, vegetable, orsynthetic origin, such as peanut oil, soybean oil, mineral oil, sesameoil, and the like. Water is an exemplary carrier when a formulation isadministered intravenously. Saline solutions, blood plasma medium,aqueous dextrose, and glycerol solutions can also be employed as liquidcarriers, particularly for injectable solutions. Oral formulations caninclude, without limitation, pharmaceutical grades of mannitol, lactose,starch, magnesium stearate, sodium saccharine, cellulose, magnesiumcarbonate, and the like. A more complete explanation of parenteralpharmaceutical excipients can be found in Remington, The Science andPractice of Pharmacy, 19th Edition, Philadelphia, Pa.:LippincottWilliams & Wilkins, 1995, Chapter 95. Excipients may also include, forexample, pharmaceutically acceptable salts to adjust the osmoticpressure, lipid carriers such as cyclodextrins, proteins such as serumalbumin, hydrophilic agents such as methyl cellulose, detergents,buffers, preservatives and the like. Other examples of pharmaceuticalexcipients include starch, glucose, lactose, sucrose, gelatin, malt,rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate,talc, sodium chloride, dried skim milk, glycerol, propylene, glycol,water, ethanol, and the like. A formulation, if desired, can alsocontain minor amounts of wetting or emulsifying agents, or pH bufferingagents.

A dosage regimen utilizing a PPARδ agonist is selected in accordancewith a variety of factors including type, species, age, weight, sex andphysical condition of the subject; the route of administration; and/orthe particular PPARδ agonist formulation employed. An ordinarily skilledphysician or veterinarian can readily determine an effective amount of aPPARδ agonist (or formulation thereof) useful for enhancing an exerciseeffect in a subject.

In some method embodiments involving oral administration, oral dosagesof a PPARδ agonist will generally range between about 0.001 mg per kg ofbody weight per day (mg/kg/day) to about 100 mg/kg/day, and such asabout 0.01-10 mg/kg/day (unless specified otherwise, amounts of activeingredients are on the basis of a neutral molecule, which may be a freeacid or free base). For example, an 80 kg subject would receive betweenabout 0.08 mg/day and 8 g/day, such as between about 0.8 mg/day and 800mg/day. A suitably prepared medicament for once a day administrationwould thus contain between 0.08 mg and 8 g, such as between 0.8 mg and800 mg. In some instance, formulation including a PPARδ agonist may beadministered in divided doses of two, three, or four times daily. Foradministration twice a day, a suitably prepared medicament as describedabove would contain between 0.04 mg and 4 g, such as between 0.4 mg and400 mg. Dosages outside of the aforementioned ranges may be necessary insome cases. Examples of daily dosages that may be given in the range of0.08 mg to 8 g per day include 0.1 mg, 0.5 mg, 1 mg, 2.5 mg, 5 mg, 10mg, 25 mg, 50 mg, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 800mg, 1 g, 2 g, 4 g and 8 g. These amounts can be divided into smallerdoses if administered more than once per day (e.g., one-half the amountin each administration if the drug is taken twice daily).

For some method embodiments involving administration by injection (e.g.,intravenously or subcutaneous injection), a subject would receive aninjected amount that would deliver the active ingredient inapproximately the quantities described above. The quantities may beadjusted to account for differences in delivery efficiency that resultfrom injected drug forms bypassing the digestive system. Such quantitiesmay be administered in a number of suitable ways, e.g. large volumes oflow concentrations of active ingredient during one extended period oftime or several times a day, low volumes of high concentrations ofactive ingredient during a short period of time, e.g. once a day.Typically, a conventional intravenous formulation may be prepared whichcontains a concentration of active ingredient of between about 0.01-1.0mg/ml, such as for example 0.1 mg/ml, 0.3 mg/ml, or 0.6 mg/ml, andadministered in amounts per day equivalent to the amounts per day statedabove. For example, an 80 kg subject, receiving 8 ml twice a day of anintravenous formulation having a concentration of active ingredient of0.5 mg/ml, receives 8 mg of active ingredient per day.

In other method embodiments, a PPARδ agonist (or a formulation thereof)can be administered at about the same dose throughout a treatmentperiod, in an escalating dose regimen, or in a loading-dose regime (forexample, in which the loading dose is about two to five times amaintenance dose). In some embodiments, the dose is varied during thecourse of PPARδ agonist usage based on the condition of the subjectreceiving the composition, the apparent response to the composition,and/or other factors as judged by one of ordinary skill in the art. Insome embodiments long-term administration of a PPARδ agonist (orformulation thereof) is contemplated, for instance in order to effectsustained enhancement of an exercise effect (such as aerobic endurance,e.g., running endurance).

IV. Methods for Determining Drug-Induced Enhancement of ExercisePerformance

The use of performance-enhancing substances (PES), particularly bychildren and professional athletes, has been in the news because ofpotential adverse health consequences and the arguable effects that suchpractices have on moral development of the individual and on fairathletic competition for all (Committee on Sports Medicine and Fitness,Reginald L. Washington, Md., Chairperson, Pediatrics, 115(4):1103-1106,2005). One of the discoveries provided herein is that certain genes(and/or the proteins encoded thereby) are uniquely regulated by acombination of exercise and a pharmaceutical agent (a PPARδ agonist)that results in enhanced physical performance (see Table 2). In somecases, the particular genes (and/or proteins encoded thereby) were up-or down-regulated by the combined treatment but were not affected byeither intervention alone. In other cases, the particular genes (and/orproteins encoded thereby) were not affected by the combined treatmentbut were up- or down-regulated by one or both intervention whenpracticed alone. The unique regulation of these genes (and/or theencoded proteins) makes them useful markers (either alone or in anycombination) for identifying exercising subjects who are taking (orreceiving) PES.

A PES is any substance taken in nonpharmacologic doses specifically forthe purpose of improving sports performance (e.g., by increasingstrength, power, speed, or endurance (ergogenic) or by altering bodyweight or body composition). Exemplary PES include the following: (i)pharmacologic agents (prescription or nonprescription) taken in dosesthat exceed the recommended therapeutic dose or taken when thetherapeutic indication(s) are not present (e.g., using decongestants forstimulant effect, using bronchodilators when exercise-inducedbronchospasm is not present, increasing baseline methylphenidatehydrochloride dose for athletic competition); (ii) agents used forweight control, including stimulants, diet pills, diuretics, andlaxatives, when the user is in a sport that has weight classificationsor that rewards leanness; (iii) agents used for weight gain, includingover-the-counter products advertised as promoting increased muscle mass;(iv) physiologic agents or other strategies used to enhanceoxygen-carrying capacity, including erythropoietin and red blood celltransfusions (blood doping); (v) any substance that is used for reasonsother than to treat a documented disease state or deficiency; (vi) anysubstance that is known to mask adverse effects or detectability ofanother performance-enhancing substance, and/or (vii) nutritionalsupplements taken at supraphysiologic doses or at levels greater thanrequired to replace deficits created by a disease state, training,and/or participation in sports. In one example the PES is GW1516.

The biomarkers of substance-induced performance enhancement identifiedherein and useful in a disclosed method include one or more (or anycombination of) the genes (and/or proteins encoded thereby) listed inTable 2, and in some examples listed in Table 4. In particular methodembodiments, at least 2, at least 3, at least 5, at least 7, at least10, at least 15, at least 20, at least 30, or at least 40 of the genes(and/or proteins encoded thereby) listed in Table 2 (or Table 4) aredetected in a disclosed method. In one example at least one gene (and/orprotein encoded thereby) from each class listed in Table 2 (e.g.,cytokines, fat metabolism) is analyzed.

In more specific method embodiments, upregulated expression is detectedfor one or more of the following genes (or proteins encoded thereby):adipose differentiation related protein; stearoyl-Coenzyme A desaturase2; acetyl-Coenzyme A acetyltransferase 2; ATP citrate lyase;adiponectin, C1Q and collagen domain containing; diacylglycerolO-acyltransferase 2; lipase, hormone sensitive; monoglyceride lipase;resistin; CD36 antigen; fatty acid binding protein 4, adipocyte;lipoprotein lipase; microsomal glutathione S-transferase 1; GPI-anchoredmembrane protein 1; dual specificity phosphatase 7; homeodomaininteracting protein kinase 3; insulin-like growth factor binding protein5; protein phosphatase 2 (formerly 2A), regulatory subunit A (PR 65),beta isoform; protein tyrosine phosphatase-like (proline instead ofcatalytic arginine); member b; CCAAT/enhancer binding protein (C/EBP),alpha; nuclear receptor subfamily 1, group D, member 2(Reverb-b);transferring; archain 1; solute carrier family 1 (neutral amino acidtransporter), member 5; RIKEN cDNA 1810073N04 gene; haptoglobin; retinolbinding protein 4, plasma; phosphoenolpyruvate carboxykinase 1,cytosolic; cell death-inducing DFFA-like effector c; interferon,alpha-inducible protein 27; carbonic anhydrase 3; cysteine dioxygenase1, cytosolic; DNA segment, Chr 4, Wayne State University 53, expressed;dynein cytoplasmic 1 intermediate chain 2; Kruppel-like factor 3(basic); thyroid hormone responsive SPOT14 homolog (Rattus); cytochromeP450, family 2, subfamily e, polypeptide 1; complement factor D(adipsin); and/or transketolase. In particular method embodiments,upregulation of at least 2, at least 3, at least 5, at least 7, at least10, at least 15, at least 20, at least 30, or at least 38 of theforegoing genes (and/or proteins encoded thereby) are detected in adisclosed method.

In other method embodiments, downregulated expression is detected in oneor more of the following genes (and/or proteins encoded thereby):gamma-glutamyl carboxylase; 3-oxoacid CoA transferase 1; solute carrierfamily 38, member 4; annexin A7; CD55 antigen; RIKEN cDNA 1190002H23gene; fusion, derived from t(12; 16) malignant liposarcoma (human);lysosomal membrane glycoprotein 2; and/or neighbor of Punc E11. Inparticular method embodiments, downregulation of at least 2, at least 3,at least 5, or at least 7 of the foregoing genes (and/or proteinsencoded thereby) are detected in a disclosed method.

In still other method embodiments, a combination of upregulated genes(and/or proteins encoded thereby) and downregulated genes (and/orproteins encoded thereby) as described above is detected in a samplefrom a subject (such as, an exercised or exercise-trained subject).

Yet other method embodiments involve the detection in a sample of acombination of the above-described upregulated genes (and/or proteinsencoded thereby) and/or the above-described downregulated genes (and/orproteins encoded thereby), and/or the above-described exercise-regulatedgenes that are not affected by exercise combined with PPARδadministration.

Disclosed methods may be used for detecting PES use in any subjectcapable of taking or receiving one or more such PES. In some methodembodiments, a subject is a living multi-cellular vertebrate organism(e.g., human and/or non-human animals). In other exemplary methods, asubject is a mammal (including humans and/or non-human mammals) or, inmore particular examples, a racing mammal (such as a horse, a dog, or ahuman). In still other methods, a subject is an exercise-trainedsubject. Some representative exercised-trained subjects have performedphysical activity (such described in detail above) for at least 4 weeks,for at least 6 weeks, for at least 3 months, or for at least 6 months.Other exercise-trained subjects may be student athletes and/orprofessional athletes (including, in some examples, non-humanprofessional athletes, such as race horses and/or racing dogs).

Any sample from a subject (e.g., a biological sample) in which can bedetected one or more genes and/or proteins uniquely regulated byexercise in combination with PPARδ agonist intake (as described indetail throughout this specification) is contemplated for use in adisclosed method. Exemplary samples for use in a disclosed methodinclude blood, saliva, urine, muscle biopsy (e.g., skeletal musclebiopsy), cheek swab, fecal sample, sweat, and/or sperm.

Methods of detecting the expression of genes and/or proteins in a sample(e.g, biological sample) are very well known (see, e.g., U.S. Pat. Nos.6,911,307; 6,893,824; 5,972,692; 5,972,602; 5,776,672; 7,031,847;6,816,790; 6,811,977; 6,806,049; 6,203,988; and/or 6,090,556).

In particular method embodiments, expression of one or more genesidentified herein can be detected by any method of nucleic acidamplification (such as, polymerase chain reaction (PCR) or anyadaptation thereof, ligase chain reaction, transcription-basedamplification systems, cycling probe reaction, Qβ replicaseamplification, strand displacement amplification, and/or rolling circleamplification), solid-surface hybridization assays (such as Northernblot, dot blot, gene chips, and/or reversible target capture), solutionhybridization assays (such as MAP technology (which uses a liquidsuspension array of 100 sets of 5.5 micron probe-conjugated beads, eachinternally dyed with different ratios of two spectrally distinctfluorophores to assign it a unique spectral address)), and/or in situhybridization. Various of the foregoing nucleic acid detection methodsare described in detail in the review by Wolcott (Clin. Microbiol. Rev.,5(4):370-386, 1992). Other detailed and long-established protocols forpracticing some such nucleic acid detection methods are found inSambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition,Cold Spring Harbor Laboratory Press, 1989; Sambrook et al., MolecularCloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor Press,2001; Ausubel et al., Current Protocols in Molecular Biology, GreenePublishing Associates, 1992 (and Supplements to 2000); and/or Ausubel etal., Short Protocols in Molecular Biology: A Compendium of Methods fromCurrent Protocols in Molecular Biology, 4th edition, Wiley & Sons, 1999.

In other method embodiments, expression of one or more proteins encodedby corresponding genes identified herein can be detected by Westernblot, immunohistochemistry, immunoprecipitation, antibody microarrays,ELISA, and/or by functional assay (e.g., kinase assay, ATPase assay,substrate (or ligand) binding assay, protein-protein binding assay, orother assay suitable for measuring a particular protein function).

If the pattern of expression identified in the test subject is similarto that shown in Table 2 (e.g., the genes shown as upregulated anddownregulated in Table 2 are observed in the subject to be upregulatedand downregulated, respectively), this indicates that the subject istaking a PES, such as a PPARδ agonist (e.g., GW1516). In contrast, Ifthe pattern of expression identified in the test subject is different tothat shown in Table 2 (e.g., the genes shown as upregulated anddownregulated in Table 2 are observed in the subject to be notdifferentially expressed or show a different pattern of regulation),this indicates that the subject is not taking a PES, such as a PPARδagonist (e.g., GW1516).

V. Methods for Identifying Agents of Potential Interest

This disclosure identifies a previously unknown protein-proteininteraction between PPARδ and particular exercise-induced kinases (e.g.,AMPK, such as the AMPKα1 and/or AMPKα2 subunit(s) of AMPK). Theinteraction between PPARδ and AMPK may have important functionaloutcomes, such as enhancing exercise performance (e.g., aerobic exerciseperformance, such as running endurance) in a subject.

The foregoing discoveries enable methods for identify agents, e.g.,having potential to enhance exercise performance (e.g., aerobic exerciseperformance, such as running endurance) in a subject. In some suchmethods, agents that affect (e.g., enhance, weaken, or substantiallydisrupt) the protein-protein interaction are identified. In other suchmethods, agents that affect (e.g., increase, decrease, or substantiallyeliminate) AMPK-dependent phosphorylation of a PPARδ complex areidentified.

A. Exemplary Agents

An “agent” is any substance or any combination of substances that isuseful for achieving an end or result; for example, a substance orcombination of substances useful for modulating a protein activity(e.g., AMPK-dependent phosphorylation of a PPARδ complex), or useful formodifying or affecting a protein-protein interaction (e.g., PPARδ-AMPKinteraction). Any agent that has potential (whether or not ultimatelyrealized) to modulate any aspect of the PPARδ-AMPK interaction disclosedherein is contemplated for use in the screening methods of thisdisclosure.

Exemplary agents include, but are not limited to, peptides such as, forexample, soluble peptides, including but not limited to members ofrandom peptide libraries (see, e.g., Lam et al., Nature, 354:82-84,1991; Houghten et al., Nature, 354:84-86, 1991), and combinatorialchemistry-derived molecular library made of D- and/or L-configurationamino acids, phosphopeptides (including, but not limited to, members ofrandom or partially degenerate, directed phosphopeptide libraries; see,e.g., Songyang et al., Cell, 72:767-778, 1993), antibodies (including,but not limited to, polyclonal, monoclonal, humanized, anti-idiotypic,chimeric or single chain antibodies, and Fab, F(ab′)₂ and Fab expressionlibrary fragments, and epitope-binding fragments thereof), small organicor inorganic molecules (such as, so-called natural products or membersof chemical combinatorial libraries), molecular complexes (such asprotein complexes), or nucleic acids.

Libraries (such as combinatorial chemical libraries) useful in thedisclosed methods include, but are not limited to, peptide libraries(see, e.g., U.S. Pat. No. 5,010,175; Furka, Int. J. Pept. Prot. Res.,37:487-493, 1991; Houghton et al., Nature, 354:84-88, 1991; PCTPublication No. WO 91/19735), encoded peptides (e.g., PCT Publication WO93/20242), random bio-oligomers (e.g., PCT Publication No. WO 92/00091),benzodiazepines (e.g., U.S. Pat. No. 5,288,514), diversomers such ashydantoins, benzodiazepines and dipeptides (Hobbs et al., Proc. Natl.Acad. Sci. USA, 90:6909-6913, 1993), vinylogous polypeptides (Hagiharaet al., J. Am. Chem. Soc., 114:6568, 1992), nonpeptidal peptidomimeticswith glucose scaffolding (Hirschmann et al., J. Am. Chem. Soc.,114:9217-9218, 1992), analogous organic syntheses of small compoundlibraries (Chen et al., J. Am. Chem. Soc., 116:2661, 1994),oligocarbamates (Cho et al., Science, 261:1303, 1003), and/or peptidylphosphonates (Campbell et al., J. Org. Chem., 59:658, 1994), nucleicacid libraries (see Sambrook et al. Molecular Cloning, A LaboratoryManual, Cold Springs Harbor Press, N.Y., 1989; Ausubel et al., CurrentProtocols in Molecular Biology, Green Publishing Associates and WileyInterscience, N.Y., 1989), peptide nucleic acid libraries (see, e.g.,U.S. Pat. No. 5,539,083), antibody libraries (see, e.g., Vaughn et al.,Nat. Biotechnol., 14:309-314, 1996; PCT App. No. PCT/US96/10287),carbohydrate libraries (see, e.g., Liang et al., Science, 274:1520-1522,1996; U.S. Pat. No. 5,593,853), small organic molecule libraries (see,e.g., benzodiazepines, Baum, C&EN, January 18, page 33, 1993;isoprenoids, U.S. Pat. No. 5,569,588; thiazolidionones andmethathiazones, U.S. Pat. No. 5,549,974; pyrrolidines, U.S. Pat. Nos.5,525,735 and 5,519,134; morpholino compounds, U.S. Pat. No. 5,506,337;benzodiazepines, U.S. Pat. No. 5,288,514) and the like.

Libraries useful for the disclosed screening methods can be produce in avariety of manners including, but not limited to, spatially arrayedmultipin peptide synthesis (Geysen, et al., Proc. Natl. Acad. Sci.,81(13):3998-4002, 1984), “tea bag” peptide synthesis (Houghten, Proc.Natl. Acad. Sci., 82(15):5131-5135, 1985), phage display (Scott andSmith, Science, 249:386-390, 1990), spot or disc synthesis (Dittrich etal., Bioorg. Med. Chem. Lett., 8(17):2351-2356, 1998), or split and mixsolid phase synthesis on beads (Furka et al., Int. J. Pept. ProteinRes., 37(6):487-493, 1991; Lam et al., Chem. Rev., 97(2):411-448, 1997).Libraries may include a varying number of compositions (members), suchas up to about 100 members, such as up to about 1000 members, such as upto about 5000 members, such as up to about 10,000 members, such as up toabout 100,000 members, such as up to about 500,000 members, or even morethan 500,000 members.

In one convenient embodiment, high throughput screening methods involveproviding a combinatorial chemical or peptide library containing a largenumber of potential therapeutic compounds (e.g., affectors of AMPK-PPARδprotein-protein interactions). Such combinatorial libraries are thenscreened in one or more assays as described herein to identify thoselibrary members (particularly chemical species or subclasses) thatdisplay a desired characteristic activity (such as increasing ordecreasing an AMPK-PPARδ protein-protein interaction). The compoundsthus identified can serve as conventional “lead compounds” or canthemselves be used as potential or actual therapeutics. In someinstances, pools of candidate agents may be identify and furtherscreened to determine which individual or subpools of agents in thecollective have a desired activity.

B. Exemplary Assays

As disclosed herein, PPARδ forms a protein-protein interaction with AMPKor one or more of its subunits (such as AMPKα1 and/or AMPKα2). Agentsthat affect (e.g., increase or decrease) an AMPK-PPARδ interaction orAMP-dependent phosphorylation of a PPARδ complex may have the effect ofenhancing exercise performance (e.g., aerobic exercise performance, suchas running endurance) in a subject and, therefore, are desirable toidentify.

In screening methods described here, tissue samples, isolated cells,isolated polypeptides, and/or test agents can be presented in a mannersuitable for high-throughput screening; for example, one or a pluralityof isolated tissue samples, isolated cells, or isolated polypeptides canbe inserted into wells of a microtitre plate, and one or a plurality oftest agents can be added to the wells of the microtitre plate.Alternatively, one or a plurality of test agents can be presented in ahigh-throughput format, such as in wells of microtitre plate (either insolution or adhered to the surface of the plate), and contacted with oneor a plurality of isolated tissue samples, isolated cells, and/orisolated polypeptides under conditions that, at least, sustain thetissue sample or isolated cells or a desired polypeptide function and/orstructure. Test agents can be added to tissue samples, isolated cells,or isolated polypeptides at any concentration that is not lethal totissues or cells, or does not have an adverse effect on polypeptidestructure and/or function. It is expected that different test agentswill have different effective concentrations. Thus, in some methods, itis advantageous to test a range of test agent concentrations.

Disclosed methods envision, as appropriate, the use of PPARδ or AMPK(such as AMPKα1 or AMPKα2) or functional fragments of any thereof ascontained, independently, in a subject, one or a plurality of cells orcellular extracts, one or a plurality of tissue or tissue extracts, oras an isolated polypeptide. PPARδ ligand optionally is included (or isomitted) in disclosed methods.

1. Agents that Affect a Protein-Protein Interaction

A “direct association” between two or more polypeptides (such as, PPARδand AMPK (such as AMPKα1 or AMPKα2) is characterized by physical contactbetween at least a portion of the interacting polypeptides that is ofsufficient affinity and specificity that, for example,immunoprecipitation of one of the polypeptides also will specificallyprecipitate the other polypeptide; provided that the immunoprecipitatingantibody does not also affect the site(s) involved in the interaction. Adirect association between polypeptides also may be referred to as a“protein-protein interaction.” The binding of one polypeptide to anotherin a protein-protein interaction (e.g., PPARδ to AMPK (or AMPKα1 and/orAMPKα2) and vice versa) is considered “specific binding”.

Agents that affect an AMPK-PPARδ interaction can be identified by avariety of assays, including solid-phase or solution-based assays. In anexemplary solid-phase assay, PPARδ or an AMPK-binding fragment thereofand AMPK or a subunit thereof (such as AMPKα1 and/or AMPKα2) or aPPARδ-binding fragment thereof are mixed under conditions in which PPARδand AMPK (or its subunit(s) or functional fragments) normally interact(e.g., co-immunoprecipitate). One of the binding partners is labeledwith a marker such as biotin, fluoroscein, EGFP, or enzymes to alloweasy detection of the labeled component. The unlabeled binding partneris adsorbed to a support, such as a microtiter well or beads. Then, thelabeled binding partner is added to the environment where the unlabeledbinding partner is immobilized under conditions suitable for interactionbetween the two binding partners. One or more test compounds, such ascompounds in one or more of the above-described libraries, areseparately added to individual microenvironments containing theinteracting binding partners. Agents capable of affecting theinteraction between the binding partners are identified, for instance,as those that increase or decrease (e.g., increase) retention or bindingof the signal (i.e., labeled binding partner) in the reactionmicroenvironment, for example, in a microtiter well or on a bead forexample. As discussed previously, combinations of agents can beevaluated in an initial screen to identify pools of agents to be testedindividually, and this process is easily automated with currentlyavailable technology.

In other method embodiments, solution phase selection can be used toscreen large complex libraries for agents that specifically affectprotein-protein interactions (see, e.g., Boger et al., Bioorg. Med.Chem. Lett., 8(17):2339-2344, 1998); Berg et al., Proc. Natl. Acad.Sci., 99(6):3830-3835, 2002). In one such example, each of two proteinsthat are capable of physical interaction (for example, PPARδ (orAMPK-binding fragments thereof) and AMPK or AMPKα1 or AMPKα2 (orPPARδ-binding fragments of any thereof) are labeled with fluorescent dyemolecule tags with different emission spectra and overlapping adsorptionspectra. When these protein components are separate, the emissionspectrum for each component is distinct and can be measured. When theprotein components interact, fluorescence resonance energy transfer(FRET) occurs resulting in the transfer of energy from a donor dyemolecule to an acceptor dye molecule without emission of a photon. Theacceptor dye molecule alone emits photons (light) of a characteristicwavelength. Therefore, FRET allows one to determine the kinetics of twointeracting molecules based on the emission spectra of the sample. Usingthis system, two labeled protein components are added under conditionswhere their interaction resulting in FRET emission spectra. Then, one ormore test compounds, such as compounds in one or more of theabove-described libraries, are added to the environment of the twolabeled protein component mixture and emission spectra are measured. Anincrease in the FRET emission, with a concurrent decrease in theemission spectra of the separated components indicates that an agent (orpool of candidate agents) has affected (e.g., enhanced) the interactionbetween the protein components.

Interactions between PPARδ (or AMPK-binding fragments thereof) and AMPKor AMPKα1 or AMPKα2 (or PPARδ-binding fragments of any thereof) also canbe determined (e.g., quantified) by co-immunoprecipitation of therelevant component polypeptides (e.g., from cellular extracts), byGST-pull down assay (e.g., using purified GST-tagged bacterialproteins), and/or by yeast two-hybrid assay, each of which methods isstandard in the art. Conducting any one or more such assays in thepresence and, optionally, absence of a test compound can be used toidentify agents that improve or enhance (or, in other embodiments,decrease or inhibit) the interaction between PPARδ (or AMPK-bindingfragments thereof) and AMPK or AMPKα1 or AMPKα2 (or PPARδ-bindingfragments of any thereof) in the presence of a test compound as comparedto in the absence of the test compound or as compared to some otherstandard or control.

In certain method embodiments, one or more AMPK (such as AMPKα1 and/orAMPKα2)-binding fragments of PPARδ and/or one or more PPARδ-bindingfragments of AMPK (such as AMPKα1 and/or AMPKα2) are used. Polypeptidefragments having the desired binding activities can be identified bymaking a series of defined PPARδ fragments and/or AMPK (such as AMPKα1or AMPKα2) fragments using methods standard in the art. For example,cDNA encoding the protein(s) of interest (e.g., PPARδ or AMPK) can beserially truncated from the 3′ or 5′ end (provided that a start codon isengineered into 5′ truncations) using conveniently located restrictionenzyme sites (or other methods) and leaving intact (or otherwisecorrecting) the proper reading frame. Conveniently, a nucleic acidsequence encoding an epitope tag (such as a FLAG tag) is placed in framewith (and substantially adjacent to) the truncated protein-encodingsequence to produce a nucleic acid sequence encoding an epitope-taggedprotein fragment. The epitope-tagged protein fragment can be expressedin any convenient expression system (such as a bacterial expressionsystem), isolated or not, and mixed with a sample containing a proteinor other protein fragment to which the epitope-tagged protein fragmentmay bind. An antibody specific for the tag (or other region of theprotein fragment) can be used to immunoprecipitate the fragment ofinterest together with any protein(s) or protein fragment(s) that bindto it. Protein(s) or protein fragment(s) that bind to the epitope-taggedprotein fragment of interest can be particular identified, e.g., byWestern blot.

In particular methods, the formation of a PPARδ-AMPK (such as AMPKα1and/or AMPKα2) complex (including complexes including one or both ofPPARδ-binding AMPK fragments and/or AMPK-binding PPARδ fragments) or theaffinity of PPARδ (or AMPK-binding fragments thereof) and AMPK (orPPARδ-binding fragments thereof) for each other is increased when theamount of such complex or the binding affinity is at least 5%, at least10%, at least 20%, at least 30%, at least 50%, at least 100% or at least250% higher than a control measurement (e.g., in the same test systemprior to addition of a test agent, or in a comparable test system in theabsence of a test agent).

In other particular methods, the formation of a PPARδ-AMPK (such asAMPKα1 and/or AMPKα2) complex (including complexes including one or bothof PPARδ-binding AMPK fragments and/or AMPK-binding PPARδ fragments) orthe affinity of PPARδ (or AMPK-binding fragments thereof) and AMPK (orPPARδ-binding fragments thereof) for each other is decreased when theamount of such complex or the binding affinity is at least 5%, at least10%, at least 20%, at least 30%, at least 50%, at least 100% or at least250% lower than a control measurement (e.g., in the same test systemprior to addition of a test agent, or in a comparable test system in theabsence of a test agent).

2. Agents that Affect AMPK-Dependent Phosphorylation

Disclosed are methods of screening test agents for those that affect(e.g., increase or decrease) AMPK (e.g., AMPKα1 and/or AMPKα2)-dependentphosphorylation of the PPARδ complex. Agents that affect AMPK-dependentphosphorylation of the PPARδ complex can be identified by a variety ofassays, such adaptations of solid-phase- or solution-based assaysdescribed above, where the end point to be detected is phosphorylationof one or more components of the PPARδ complex.

Methods for detecting protein phosphorylation are conventional (see,e.g., Gloffke, The Scientist, 16(19):52, 2002; Screaton et al., Cell,119:61-74, 2004) and detection kits are available from a variety ofcommercial sources (see, e.g., Upstate (Charlottesville, Va., USA),Bio-Rad (Hercules, Calif., USA), Marligen Biosciences, Inc. (Ijamsville,Md., USA), Calbiochem (San Diego, Calif., USA). Briefly, phosphorylatedprotein (e.g., phosphorylation of one or more components of the PPARδcomplex) can be detected using stains specific for phosphorylatedproteins in gels. Alternatively, antibodies specific phosphorylatedproteins can be made or commercially obtained. Antibodies specific forphosphorylated proteins can be, among other things, tethered to thebeads (including beads having a particular color signature) or used inELISA or Western blot assays.

In one example, a PPARδ complex (or a fragment thereof containing anAMPK phosphorylation site) and AMPK or one or more of it subunits (suchas AMPKα1 and/or AMPKα2) or functional fragments thereof that arecapable of phosphorylation are mixed under conditions whereby a PPARδcomplex is phosphorylated by AMPK. A PPARδ complex is adsorbed to asupport, such as a microtiter well or beads. Then, AMPK (or its one ormore subunits (such as AMPKα1 and/or AMPKα2) or phosphorylation-capablefragments thereof) is added to the environment where the complex isimmobilized. A phosphate donor typically is also included in theenvironment. The phosphate to be donated, optionally, can be labeled.One or more test compounds, such as compounds in one or more of theabove-described libraries, are separately added to the individualmicroenvironments. Agents capable of affecting AMPK-dependentphosphorylation are identified, for instance, as those that enhance (orinhibit) phosphorylation of immobilized PPARδ complex. In embodimentsinvolving a labeled phosphate donor, phosphorylation of immobilizedPPARδ complex can be determined by retention or binding of a labeledphosphate in the reaction microenvironment, for example, in a microtiterwell or on a bead for example. In other embodiments, such reactions cantake place in solution (i.e., with no immobilized components), PPARδcomplex can be isolated from the solution (e.g., by immunoprecipitationwith PPARδ-specific or phosphate-specific antibodies), and its level ofphosphorylation in the presence (and, optionally, absence) of one ofmore test agents determined as previously discussed.

In particular methods, the phosphorylation of a PPARδ complex isincreased when such posttranslational modification is detectablymeasured or when such posttranslational modification is at least 20%, atleast 30%, at least 50%, at least 100% or at least 250% higher thancontrol measurements (e.g., in the same test system prior to addition ofa test agent, or in a comparable test system in the absence of a testagent, or in a comparable test system in the absence of AMPK).

In particular methods, the phosphorylation of PPARδ complex is decreasedwhen such posttranslational modification is detectably reduced or whensuch posttranslational modification is at least 20%, at least 30%, atleast 50%, at least 100% or at least 250% lower than controlmeasurements (e.g., in the same test system prior to addition of a testagent, or in a comparable test system in the absence of a test agent, orin a comparable test system in the absence of AMPK).

C. Screening Assay Target(s)

1. PPARδ

A PPARδ polypeptide useful in a disclosed screening method is any knownPPARδ receptor. Also useful in the disclosed screening methods arehomologs, functional fragments, or functional variants of a PPARδ thatretains at least AMPK-binding activity as described herein for aprototypical PPARδ polypeptide (see Example 6).

The amino acid sequences of prototypical PPARδ polypeptides (andPPARδ-encoding nucleic acid sequences) are well known. Exemplary PPARδamino acid sequences and PPARδ-encoding nucleic acid sequences aredescribed, for instance, in U.S. Pat. No. 5,861,274, and U.S. Pat. Appl.Pub. No. 20060154335 (each of which is expressly incorporated herein byreference), and in GenBank Accession Nos. NP_(—)035275 (SEQ ID NO:1)(GI:33859590) (Mus musculus amino acid sequence); NM_(—)011145.3 (SEQ IDNO:2) (GI:89001112) (Mus musculus nucleic acid sequence); NP_(—)006229(SEQ ID NO:3) (GI:5453940) (Homo sapiens amino acid sequence);NM_(—)006238.3 (SEQ ID NO:4) (GI:89886454) (Homo sapiens nucleic acidsequence); NP_(—)037273 (SEQ ID NO:5) (GI:6981384) (Rattus norvegicusamino acid sequence); NM_(—)013141.1 (SEQ ID NO:6) (GI:6981383) (Rattusnorvegicus nucleic acid sequence); NP_(—)990059 (SEQ ID NO:7)(GI:45382025) (Gallus gallus amino acid sequence) or NM_(—)204728.1 (SEQID NO:8) (GI:45382024) (Gallus gallus nucleic acid sequence). In somemethod embodiments, a PPARδ homolog or functional variant shares atleast 60% amino acid sequence identity with a prototypical PPARδpolypeptide; for example, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, or at least 98% amino acid sequence identitywith an amino acid sequence as set forth in U.S. Pat. No. 5,861,274,U.S. Pat. Appl. Pub. No. 20060154335, or GenBank Accession No.NP_(—)035275 (SEQ ID NO:1) (GI:33859590) (Mus musculus amino acidsequence); NP_(—)006229 (SEQ ID NO:3) (GI:5453940) (Homo sapiens aminoacid sequence); NP_(—)037273 (SEQ ID NO:5) (GI:6981384) (Rattusnorvegicus amino acid sequence); or NP_(—)990059 (SEQ ID NO:7)(GI:45382025) (Gallus gallus amino acid sequence). In other methodembodiments, a PPARδ homolog or functional variant has one or moreconservative amino acid substitutions as compared to with a prototypicalPPARδ polypeptide; for example, no more than 3, 5, 10, 15, 20, 25, 30,40, or 50 conservative amino acid changes compared to an amino acidsequence as set forth in U.S. Pat. No. 5,861,274, U.S. Pat. Appl. Pub.No. 20060154335, or GenBank Accession No. NP_(—)035275 (SEQ ID NO:1)(GI:33859590) (Mus musculus amino acid sequence); NP_(—)006229 (SEQ IDNO:3) (GI:5453940) (Homo sapiens amino acid sequence); NP_(—)037273 (SEQID NO:5) (GI:6981384) (Rattus norvegicus amino acid sequence); orNP_(—)990059 (SEQ ID NO:7) (GI:45382025) (Gallus gallus amino acidsequence). The following table shows exemplary conservative amino acidsubstitutions:

Conservative Original Residue Substitutions Ala Ser Arg Lys Asn Gln; HisAsp Glu Cys Ser Gln Asn Glu Asp Gly Pro His Asn; Gln Ile Leu; Val LeuIle; Val Lys Arg; Gln; Glu Met Leu; Ile Phe Met; Leu; Tyr Ser Thr ThrSer Trp Tyr Tyr Trp; Phe Val Ile; Leu

Some method embodiments involve a PPARδ functional fragment (such as anAMPK-binding fragment), which can be any portion of a full-length knownPPARδ polypeptide, including, e.g., about 20, about 30, about 40, about50, about 75, about 100, about 150 or about 200 contiguous amino acidresidues of same; provided that the fragment retains a PPARδ function ofinterest (e.g., AMPK binding). PPARδ encompasses known functional motifs(such as ligand-binding domain, a DNA-binding domain, and atransactivation domain).

2. AMPK

Mammalian AMP-activated kinase (AMPK) is a heterotrimeric proteincomposed of 1 alpha subunit, 1 beta subunit, and 1 gamma subunit. Thereare, at least, two known isoforms of the alpha subunit (α1 and α2).AMPKα1 and AMPKα2 have 90% amino acid sequence identity within theircatalytic cores but only 61% in their C-terminal tails (see OnlineMendelian Inheritance in Man (OMIM) Database Accession No. 602739;publicly available at the following website:ncbi.nlm.nih.gov/entrez/dispomim.cgi?id=602739).

An AMPK (such as AMPKα1 and/or AMPKα2) polypeptide useful in a disclosedscreening method is any known AMPK protein or subunit thereof (such asAMPKα1 and/or AMPKα2). Also useful in the disclosed screening methodsare homologs, functional fragments, or functional variants of an AMPKprotein or subunit thereof (such as AMPKα1 and/or AMPKα2) that retainsat least PPARδ-binding activity as described herein (see Example 6).

The amino acid sequences of prototypical AMPK subunits (such as AMPKα1and/or AMPKα2) (and nucleic acids sequences encoding prototypical AMPKsubunits (such as AMPKα1 and/or AMPKα2)) are well known. ExemplaryAMPKα1 amino acid sequences and the corresponding nucleic acid sequencesare described, for instance, in GenBank Accession Nos. NM_(—)206907.3(SEQ ID NO:9) (GI:94557298) (Homo sapiens transcript variant 2 REFSEQincluding amino acid and nucleic acid sequences); NM_(—)006251.5 (SEQ IDNO:10) (GI:94557300) (Homo sapiens transcript variant 1 REFSEQ includingamino acid and nucleic acid sequences); NM_(—)001013367.3 (SEQ ID NO:11)(GI:94681060) (Mus musculus REFSEQ including amino acid and nucleic acidsequences); NM_(—)001039603.1 (SEQ ID NO:12) (GI:88853844) (Gallusgallus REFSEQ including amino acid and nucleic acid sequences); andNM_(—)019142.1 (SEQ ID NO:13) (GI:11862979) (Rattus norvegicus REFSEQincluding amino acid and nucleic acid sequences). Exemplary AMPKα2 aminoacid sequences and the corresponding nucleic acid sequences aredescribed, for instance, in GenBank Accession Nos. NM_(—)006252.2 (SEQID NO:14) (GI:46877067) (Homo sapiens REFSEQ including amino acid andnucleic acid sequences); NM_(—)178143.1 (SEQ ID NO:15) (GI:54792085)(Mus musculus REFSEQ including amino acid and nucleic acid sequences);NM_(—)001039605.1 (SEQ ID NO:16) (GI:88853850) (Gallus gallus REFSEQincluding amino acid and nucleic acid sequences); and NM_(—)214266.1(SEQ ID NO:17) (GI:47523597) (Sus scrofa REFSEQ including amino acid andnucleic acid sequences).

In some method embodiments, a homolog or functional variant of an AMPKsubunit shares at least 60% amino acid sequence identity with aprototypical AMPKα1 and/or AMPKα2 polypeptide; for example, at least75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least98% amino acid sequence identity with an amino acid sequence as setforth in the GenBank Accession Nos. NM_(—)206907.3 (SEQ ID NO:9);NM_(—)006251.5 (SEQ ID NO:10); NM_(—)001013367.3 (SEQ ID NO:11);NM_(—)001039603.1 (SEQ ID NO:12); NM_(—)019142.1 (SEQ ID NO:13);NM_(—)006252.2 (SEQ ID NO:14); NM_(—)178143.1 (SEQ ID NO:15);NM_(—)001039605.1 (SEQ ID NO:16); or NM_(—)214266.1 (SEQ ID NO:17). Inother method embodiments, a homolog or functional variant of an AMPKsubunit has one or more conservative amino acid substitutions ascompared to a prototypical AMPKα1 and/or AMPKα2 polypeptide; forexample, no more than 3, 5, 10, 15, 20, 25, 30, 40, or 50 conservativeamino acid changes compared to an amino acid sequence as set forth in asset forth in GenBank Accession Nos. NM_(—)206907.3 (SEQ ID NO:9);NM_(—)006251.5 (SEQ ID NO:10); NM_(—)001013367.3 (SEQ ID NO:11);NM_(—)001039603.1 (SEQ ID NO:12); NM_(—)019142.1 (SEQ ID NO:13);NM_(—)006252.2 (SEQ ID NO:14); NM_(—)178143.1 (SEQ ID NO:15);NM_(—)001039605.1 (SEQ ID NO:16); or NM_(—)214266.1 (SEQ ID NO:17).Exemplary conservative amino acid substitutions have been previouslydescribed herein.

Some method embodiments involve a functional fragment of AMPK or asubunit thereof (such as AMPKα1 and/or AMPKα2), including aPPARδ-binding fragment or a fragment with PPARδ phosphorylationactivity. Functional fragments of AMPK or a subunit thereof (such asAMPKα1 and/or AMPKα2) can be any portion of a full-length or intact AMPKpolypeptide complex or subunit thereof (such as AMPKα1 and/or AMPKα2),including, e.g., about 20, about 30, about 40, about 50, about 75, about100, about 150 or about 200 contiguous amino acid residues of same;provided that the fragment retains at least one AMPK (or AMPKα1 and/orAMPKα2) function of interest (e.g., PPARδ binding and/or PPARδphosphorylation activity). Protein-protein interactions between PPARδand AMPK are believed to involve, at least, an AMPKα subunit (such asAMPKα1 and/or AMPKα2). Moreover, because PPARδ specifically binds bothAMPKα1 and AMPKα2 (see Example 6), such interaction likely is mediatedby the portions of these AMPKα isoforms that share the most sequencehomology (as discussed above). Accordingly, in some method embodiments,an AMPK PPARδ-binding fragment includes a functional fragmentencompassing (or consisting of) the catalytic core domain of an alphasubunit of AMPK (such as AMPKα1 and/or AMPKα2).

EXAMPLES

The following examples are provided to illustrate certain particularfeatures and/or embodiments. These examples should not be construed tolimit the invention to the particular features or embodiments described.

Example 1 Administration of PPARδ Agonist Surprisingly does not EnhancePhysical Performance in Non-Exercised Subjects

Wang et al. previously demonstrated that skeletal muscle-specificexpression of a constitutively active form of PPARδ receptor resulted intransgenic mice with skeletal muscles that had an increased number ofslow, oxidative (type I) muscle fibers and markedly increased runningendurance (Wang et al., PLoS Biol., 2:e294, 2004). This Exampledemonstrates that administration of a PPARδ agonist (GW1516) tonon-transgenic mice also results in the expression in skeletal muscle ofsome biomarkers of oxidative metabolism. However, in unexpected contrastto the results obtained by genetic activation of the PPARδ pathway,PPARδ activation by pharmacological treatment did not modify fiber-typecomposition of skeletal muscle, nor improve running endurance innon-transgenic, sedentary (also referred to as “non-exercised” or“untrained”) mice.

Male C57B/6J mice (8 wks old) were obtained from Jackson Laboratory andhoused in the Salk Institute animal care facility. The animals wereacclimated to their surroundings for one week prior to experimentation,and had access at all times to standard mouse chow and water ad libitum.

Mice were acclimated to moderate treadmill running (10 m/min for 15 min)every other day for 1 week. After acclimation, basal running endurancewas determined by placing each mouse on a treadmill, graduallyincreasing the speed from 0 to 15 m/min, and maintaining 15 m/min untilthe mouse was exhausted. The time and distance run until exhaustion wererecorded as the basal endurance values (Week 0).

Mice then were treated once per day for 4 weeks with vehicle or thePPARδ agonist, GW1516 (5 mg/kg). Treatments were administered orally.During the treatment period, mice were housed in standard laboratorycages and received only the amount of physical activity that could behad by normal movements about such cage.

Animals were euthanized by carbon dioxide asphyxiation 72 hours afterthe final treatment. Gastrocnemius and quadriceps muscles were isolated,frozen and stored at −80° C. for future analysis. Total RNA was preparedfrom quadriceps muscle using TRIZOL™ reagent (Invitrogen, Calsbad,Calif., USA) in conformance with manufacturer's instructions. Real timequantitative PCR (QPCR) was used to determine expression levels ofuncoupling protein 3 (UCP3), muscle carnitine palmitoyl transferase I(mCPT I) and pyruvate dehydrogenase kinase 4 (PDK4) using primers knownto those of ordinary skill in the art.

As shown in FIG. 1A, four weeks of GW1516 treatment induced theexpression of UCP3, mCPT I, and PDK4, in quadriceps muscle of treatedmice (compare V to GW). These changes in gene expression were detectedas early as 4 days after treatment and with drug concentrations rangingfrom 2-5 mg/kg/day. Moreover, in the gene expression studies, maximaleffects of PPARδ activation were detected in pre-dominantly fast-twitch(quadricep and gastrocnemius) but not slow-twitch (soleus) muscles.

Using primary muscle cells cultured from wild type and PPARδ null mice(Chawla et al., Proc. Natl. Acad. Sci. USA. 100(3): 1268-73, 2003; Manet al., J. Invest. Dermatol. 2007; Rando and Blau, J. Cell. Biol.125(6): 1275-87, 1994), it was confirmed that the induction of oxidativegenes by GW1516 is mediated via activation of PPARδ in skeletal muscles(FIGS. 1B-D). Moreover, this is similar to the expression changes foundin the same gene set in muscles from mice expressing the constitutivelyactive VP16-PPARδ transgene (Wang et al., PLoS Biol., 2:e294, 2004)(FIG. 1A, see TG). Collectively, these results indicate thatpharmacological activation of PPARδ can initiate an oxidative responsein adult skeletal muscle.

Expression of biomarkers characteristic of an oxidative phenotype inskeletal muscle, typically, has been correlated with increased oxidativeperformance (e.g., increased running endurance) of such skeletal muscle.This correlation was observed, for instance, in the VP16-PPARδtransgenic mouse (Wang et al., PLoS Biol., 2:e294, 2004). For this andother reasons, it was expected that GW1516 treatment similarly wouldincrease running performance. Accordingly, to determine the functionaleffects of ligand, age and weight matched cohorts of treated and controlmice were subjected to an endurance treadmill performance test before(week 0) and after (week 5) treatment.

Following four weeks of treatment and housing in standard laboratorycages without additional exercise, the running endurance ofGW1516-treated and control mice again was determined as described above.Remarkably, and despite expectations for improvement, GW1516-treatedmice did not significantly differ from controls in either the time spentor distance run on the treadmill prior to exhaustion (FIG. 1E).Furthermore, long-term drug treatment of up to 5 months also did notchange running endurance.

These results indicate that although in non-trained adult musclepharmacological activation of PPARδ induces some transcriptionalchanges, it fails to alter either fiber type composition or endurance.In summary, pharmacologic activation of the PPARδ genetic program inadult C57B1/6J mice is insufficient to promote a measurable enhancementof treadmill endurance.

Example 2 Administration of PPARδ Agonist Remodels Skeletal Muscle inExercised-Trained Subjects

Fiber type proportions in skeletal muscle are believed to be determinedby heredity and environmental factors, such as physical activity level(Simoneau and Bouchard, FASEB J., 9(11):1091-1095, 1995; Larsson andAnsved, Muscle Nerve, 8(8):714-722, 1985). Endurance exercise trainingis known to remodel the skeletal muscle by increasing type I slow-twitchfibers, oxidative enzymes, and mitochondrial density, whichprogressively alter performance (Holloszy et al., J. Appl. Physiol.56:831-8, 1984; Booth et al., Physiol Rev. 71:541-85, 1991; Schmitt etal., Physiol. Genomics. 15:148-57, 2003; Yoshioka et al., FASEB J.17:1812-9, 2003; Mahoney et al., Phys. Med. Rehabil. Clin. N. Am.16:859-73, 2005; Mahoney et al., FASEB J. 19:1498-500, 2005; Siu et al.,J. Appl. Physiol. 97:277-85, 2004; Garnier et al., FASEB J. 19:43-52,2005; Short et al., J Appl Physiol. 99:95-102, 2005; Timmons et al.,FASEB J. 19: 750-60, 2005). This example demonstrates that PPARδ agonisttreatment influences skeletal muscle on a molecular level.

To determine whether co-administration of GW1516 in the context ofendurance exercise can enhance changes in fiber type composition andmitochondrial biogenesis, the effect of GW1516 treatment on musclefiber-type composition was determined by meta-chromatic staining ofcryo-sections of gastrocnemius as described by Wang et al. (PLoS Biol.,2:e294, 2004). Meta-chromatic staining was used, following a routinemyofibrillar ATPase reaction, to demonstrate quantitative differences inphosphate deposition among different skeletal muscle fiber types and,thereby, differentiate skeletal muscle fiber types (Doriguzzi et al.,Histochem., 79(3):289-294, 1983; Ogilvie and Feeback, Stain Technol.,65(5):231-241, 1990). In this assay, muscle fibers with high ATPaseactivity (e.g., type I (slow oxidative) muscle fibers) are darklystained.

As shown in FIG. 2A, there was no significant difference in theproportion of type I (slow, oxidative) muscle fibers in thegastrocnemius muscles of vehicle- and GW1516-treated sedentary mice. Incontrast, hindlimb muscles of VP16-PPARδ transgenic mice exhibited anincreased number of type I muscle fibers when assayed by monochromaticstaining. In trained mice, GW1516 increased the proportion of type Ifibers (by ˜38%) compared to the vehicle-treated sedentary mice (FIGS.2A and 2B). Therefore, administration of a PPARδ agonist (e.g., GW1516)alone to sedentary subjects does not significantly affect the number oftype I (slow-twitch, oxidative) muscle fibers in hindlimb muscles, butcan increase the number of type I muscle fibers in hindlimb muscles oftrained subjects.

In addition to its effects on the fiber type, exercise trainingincreased skeletal muscle mitochondrial biogenesis, which can bemeasured as a function of mitochondrial DNA expression levels usingquantitative real time PCR (QPCR). Mitochondrial DNA expression levelswere determined in muscles of V, GW, Tr, and GW+Tr subjects usingquantitative real time PCR. As shown in FIG. 2C, similar to type I fiberchanges, mitochondrial DNA expression was not changed by drug alone butwas increased by approximately 50% with the combination of exercise andGW1516 treatment (FIG. 2C). Such an increase is known to contribute toenhanced endurance capacity (e.g., Holloszy, Med. Sci. Sports 7:155-64,1975).

Slow-twitch and fast-twitch muscle fiber types also can be distinguishedby myosin isoform expression (Gauthier and Lowey, J. Cell Biol.81:10-25, 1979; Fitzsimons and Hoh, Biochem. J. 193:229-33, 1981).Myosin isoform expression in skeletal muscle adapts to variousconditions, such as changes in muscle mechanics, muscle innervation, orexercise paradigm (for review, see, e.g., Baldwin and Haddad, J. Appl.Physiol., 90(1):345-57, 2001; Baldwin and Haddad, Am. J. Phys. Med.Rehabil., 81(11 Suppl):540-51, 2002; Parry, Exerc. Sport Sci. Rev.,29(4):175-179, 2001). The effect of GW1516 administration on myosinheavy chain (MHC) expression (MHC I, MHC IIa, MHC IIb) was determined bymethods known to those of ordinary skill in the art.

GW1516 treatment in sedentary mice increased the expression of MHC I (amarker of slow-twitch, oxidative muscle fibers) and decreased theexpression of MHC IIb (a marker of fast-twitch, glycolytic musclefibers) as compared to vehicle-treated, control mice. In comparison,GW1516 treatment did not alter the expression of MHC IIa (a marker offast-twitch oxidative/glycolytic muscle fibers) in sedentary mice.Therefore, at least at the transcriptional level, the PPARδ agonist wascapable of inducing some proteins characteristic of a slow-twitch musclefiber phenotype.

In summary, expression of constitutively active PPARδ in the skeletalmuscles of VP16-PPARδ transgenic mice resulted in a “long-distancerunning phenotype” with “profound and coordinated increases in oxidativeenzymes, mitochondrial biogenesis and production of specialized type Ifiber contractile proteins-the three hallmarks of muscle fiber typeswitching” (Wang et al., PLoS Biol., 2:e294, 2004). In contrast,pharmacological activation of PPARδ in normal subjects only partiallyrecapitulated VP16-PPARδ transgenesis by regulating some metabolicgenes. Markedly, administration of a PPARδ agonist to sedentary subjectsdid not lead to a change in fiber type specification (as measured bymonochromatic staining) or enhance exercise endurance. Transgenicover-expression of activated PPARδ at birth pre-programs the nascentmyofibers to trans-differentiate into slow-twitch fibers, thus impartinga high basal endurance capacity to adult transgenic mice. In contrast,since fiber type specification is completed prior to exposure of adultsto PPARδ agonist, the potential plasticity of muscle to drug treatmentalone is virtually non-existent.

This example illustrates that the genetic or pharmacologic activation ofthe PPARδ regulatory program in skeletal muscles of adult, sedentarysubjects does not have the same outcome. The ability to geneticallymanipulate skeletal muscle specification by activation of the PPARδreceptor in a transgenic mouse from early development in the absence ofexercise is not necessarily predictive of the result ofpharmacologically activating the PPARδ program in the sedentary, normaladult. The cellular “template” for PPARδ effects on skeletal muscle isvery different in a normal subject as compared to a geneticallyengineered transgenic subject. For example, in a normal adult, musclefiber specification of individual muscle groups is already determinedand the connections between muscle fibers and spinal motor neurons areestablished prior to pharmacological activation of the PPARδ-regulatedprogram. In the transgenic mouse, the constitutively active PPARδtransgene is active all the while muscle fiber specification is beingdetermined and connections between muscle fibers and motor neurons arebeing made. In addition, the effects of activation of endogenous PPARδreceptor by a single daily dose of a PPARδ agonist, which is expect tohave a transient peak exposure followed by clearance, likely are muchdifferent from the effects of the constitutive activation of aVP16-PPARδ transgene.

Example 3 The Combination of PPARδ Agonist Treatment and ExerciseTraining Significantly Affected Fatty Acid Metabolism and Markers ofFatty Acid Oxidation

In addition to affecting the contractile apparatus of skeletal muscle,exercise training also increases skeletal muscle mitochondrial density(e.g., Freyssenet et al., Arch. Physiol. Biochem., 104(2):129-141,1996). This Example illustrates that PPARδ agonist treatment (e.g.,GW1516) in exercise-trained subjects affected fatty acid metabolism inexercised muscle.

The effects of GW1516 treatment and exercise, alone or in combination,on components of the oxidative metabolism of fatty acids were determinedby measuring gene expression levels of selective biomarkers for fattyacid β-oxidation (FAO). Male C57B/6J mice (8-10 wks old) were randomlydivided into four groups (nine per group): (i) vehicle-treated andsedentary (V), (ii) GW1516-treated and sedentary (GW), (iii)vehicle-treated and exercise trained (Tr) and (iv) GW1516-treated andexercise trained (GW+Tr). Mice in all groups were acclimated to moderatetreadmill running and basal running endurance was determined asdescribed in Example 1. Thereafter, mice in the exercise-trained groupsreceived four weeks (5 days/week) of exercise training on a treadmillinclined at 5 degrees. Intensity and time of training were graduallyincreased. At the end of four weeks, all exercise-trained mice wererunning for 50 min/day at 18 m/min. Vehicle or GW1516 was administeredto the respective exercise-treated or sedentary groups as described inExample 1. Unless otherwise noted, V, GW, Tr and GW+Tr subjectsdescribed in this and the examples below were similarly treated. At theend of the drug treatment and/or training protocol (Week 5) 6 mice pergroup were subjected to the running test. These interventions do notaffect body weight and food intake in mice. RNA was prepared real timequantitative PCR performed as described in Example 1.

Confirming the results obtained in Example 1, UCP3, mCPT I, and PDK4were upregulated by GW1516 but showed no further induction with exercise(see FIGS. 1A and 3A). Unexpectedly, a second set of genes wereidentified that showed no response to exercise or GW1516 alone but wererobustly induced by the combination. This intriguing response profileincludes a series of genes involved in the regulation of fatty acidstorage [such as steroyl-CoA-desaturase (SCD1), fatty acyl coenzyme Asynthase (FAS) and serum response element binding protein 1c (SREBP 1c)]and fatty acid uptake [such as the fatty acid transporter (FAT/CD36) andlipoprotein lipase (LPL)] adding a new set of target genes to exerciseand drug treated mice (FIGS. 3B, 3C and 6A-C).

In addition to gene expression, protein expression was determined forselective oxidative biomarkers including myoglobin, UCP3, cytochrome c(CYCS) and SCD1, using Western blotting. Protein homogenates wereprepared from quadriceps muscle, separated by SDS polyacrylamide gelelectrophoresis, transferred to blotting membrane and probed withantibodies specific for myoglobin (Dako), UCP3 (Affinity Bioreagents),cytochrome c (Santacruz) SCD1 (Santacruz), and, as a loading control,tubulin (Sigma). A robust up regulation of myoglobin, UCP3, cytochromec, and SCD1 protein expression was observed with combined exercise andGW1516 treatment in comparison to treatment with the PPARδ agonist orexercise alone (FIG. 3D).

Altered triglycerides can be used to access changes in muscle oxidativecapacity. Muscle triglyceride (mTG) content was measured as previouslydescribed (Wang et al., PLoS Biol., 2:e294, 2004) using a kit fromThermo Electron Corporation. As shown in FIG. 4, mTG content wascomparable between vehicle and GW1516-treated sedentary mice and wassubstantially increased in muscle of mice receiving only exercisetraining. In contrast, dramatic increase in triglycerides in exercisedmuscle was completely reversed in GW1516-treated exercise trained mice,indicating increased fat utilization (FIG. 4).

Gene and/or protein expression that is induced by a combination ofexercise and drug treatment (e.g., PPARδ agonist administration) but notby either input alone is believed to be a new discovery. This type ofresponse can be used to further characterize the intersection ofpharmacologic and physiologic genetic networks. For example, one or moregenes and/or proteins uniquely regulated by one or more drugs (e.g.,PPARδ agonists) and exercise can be used as markers, for instance, ofillicitly boosting performance in professional and/or amateur athletes.

Example 4 Administration of PPARδ Agonist Enhances the PhysicalPerformance of Exercise-Trained Subjects

As described in Example 1, although GW1516 treatment induces wide-spreadgenomic changes associated with oxidative metabolism, nonetheless aloneit failed to increase running endurance. This finding was unexpectedbecause it was known that constitutive activation of the PPARδ genenetwork (in the VP16-PPARδ transgenic mouse) lead to a distance-runningphenotype (familiarly, a “marathon mouse”). On the other hand, assurprisingly shown in Example 3, PPARδ agonist (e.g., GW1516) treatmentin conjunction with exercise produced an enriched remodeling programthat included a series of transcriptional and posttranslationaladaptations in the skeletal muscle. This indicates that exercisetraining serves as a trigger to unmask a set of PPARδ target genes. ThisExample provides methods used to demonstrate that administration of aPPARδ agonist (e.g., GW1516) surprisingly improves physical performancein exercised (trained) subjects.

Male C57B/6J mice (8-10 wks old) were randomly divided into four groups(nine per group): (i) vehicle-treated and sedentary (V), (ii)GW1516-treated and sedentary (GW), (iii) vehicle-treated and exercisetrained (Tr) and (iv) GW1516-treated and exercise trained (GW+Tr),acclimated to moderate treadmill running as described in Example 1, andexercise-trained as described in Example 3. At the end of the drugtreatment and/or training protocol (Week 5) 6 mice per group weresubjected to the running test.

At the end of the drug treatment and/or training protocol (Week 5),running endurance of six mice per group was determined in the samemanner as was basal running endurance. No follow-up endurance tests wereperformed on three mice in each group to confirm that changes observedin the skeletal muscle were not due to the acute run, but were relatedto the exercise training.

As shown in FIGS. 5A and 5B, the same dose and duration of GW1516treatment that failed to alter running endurance in sedentary mice, whenpaired with 4 weeks of exercise training, increases running time by 68%and running distance by 70% over vehicle-treated trained mice (FIGS. 5Aand 5B, compare Week 5). Comparison of running time and distance before(week 0) and after (week 5) exercise and drug treatment revealed a 100%increment in endurance capacity for individual mice, underscoring therobustness of the combination paradigm (FIGS. 5A and 5B). In contrast,the same exercise protocol without concurrent GW1516 treatment did notsignificantly increase running endurance in C57B1/6J mice.

Hematoxylin and eosin (H&E) staining of white adipose tissue paraffinsections was performed as previously described (Wang et al., PLoS Biol.,2:e294, 2004; Wang et al., Cell, 113:159-70, 2003). As shown in FIG. 5C,GW1516 treatment in combination with exercise produced a significant(32%) reduction in the epididymal fat to body weight ratio, which wasfurther evident in the decreased cross-sectional area of the adipocytesin the same group (FIG. 5D). Therefore, the combined effects of GW1516and exercise are not restricted to muscle.

Using the methods described in Example 2, it was also demonstrated thatthe combination of GW1516 treatment and exercise training significantlyincreased the number of type I muscle fibers in exercised muscle.However, combining GW1516 treatment with exercise did not induceadditional changes in MHC I and MHC IIb expression. Therefore, althoughorally administered PPARδ agonist (GW1516) alone is capable of inducingthe expression of at least some of the contractile proteins in thePPARδ-regulated gene network (see Example 5) the transcriptional effectobserved was not sufficient to induce a post-transcriptional change infiber-type composition as was observed by meta-chromatic staining inGW1516-treated, exercised mice.

This Example illustrates that PPARδ agonist (e.g., GW1516) treatmentunexpectedly augments the performance of aerobic exercise (e.g., runningdistance and endurance) in an exercised subject. Endurance exercise isknown to channel extra-muscular fat to muscle triglyceride stores byinducing adipose tissue lipolysis to meet increased oxidative demands(Despres et al., Metabolism, 33:235-9, 1984; Mauriege et al., Am. J.Physiol., 273:E497-506, 1997; Mader et al., Int. J. Sports Med.,22:344-9, 2001; Schmitt et al., Physiol. Genomics, 15:148-57, 2003;Schrauwen-Hinderling et al., J. Clin. Endocrinol. Metab., 88:1610-6,2003). In addition, the induction of FAO components and selectiveup-regulation of fatty acid storage and up-take components inGW1516-treated, exercised mice described in Example 3 indicate enhancedmobilization of fat as fuel in skeletal muscle. Therefore, combinedexercise and GW1516 treatment dramatically increases muscle oxidativecapacity in subjects, for example by increasing local fatty acidsynthesis and/or mobilizing fatty acid stores from adipose tissue.

This is the first demonstration of how an orally active PPARδ agonistand exercise can co-operatively re-program the muscle genome and raiseendurance limits.

Example 5 The Combination of PPARδ Agonist Treatment and ExerciseTraining Produced a Unique Gene Expression Signature in Exercised Muscle

A comprehensive study of the skeletal muscle transcriptional program inV, GW, Tr and Tr+GW mice was conducted using microarray analysis.Affymetrix™ high-density oligonucleotide array mouse genome 430A 2.0chips were used. Preparation of in vitro transcription products,oligonucleotide array hybridization, and scanning were performed inconformance with Affymetrix™-provided protocols. To minimizediscrepancies due to variables, the raw expression data were scaled byusing Affymetrix™ MICROARRAY SUITE™ 5.0 software, and pairwisecomparisons were performed. The trimmed mean signal of all probe setswas adjusted to a user-specified target signal value (200) for eacharray for global scaling. No specific exclusion criteria were applied.Additional analyses were performed using the freeware program BULLFROG 7(available on the internet Barlow-LockhartBrainMapNIMHGrant.org) and theJava-based statistical tool VAMPIRE (Hsiao et al., Bioinformatics,20:3108-3127, 2004).

Genome-wide analysis of the quadriceps muscle revealed that GW1516treatment, exercise, and the combination regulated 96, 113 and 130genes, respectively (FIG. 6). Approximately 50% of the target genesregulated by GW1516 or exercise alone were the same, demonstrating thatPPARδ activation of the gene network partially mimics exercise effectson the same network.

The 130 genes regulated by the combination of GW1516 treatment andexercise training and a classification of each such gene are shown inTable 1. The 130 regulated genes included 30 fat metabolism genes, 5oxygen carriers, 5 mitochondrial genes, 3 carbohydrate metabolism genes,15 signal transduction genes, 16 transcription genes, 10 transportgenes, 3 steroid biogenesis genes, 5 heat shock genes, 2 angiogenesisgenes, 5 proliferation and apoptosis genes, 2 cytokines, and 29 others.The majority of the genes in the exercise-trained/GW1516-treated (GW+Tr)gene signature shown in Table 1 were induced (109/130). The 109upregulated genes are shown in non-bold font in Table 1 (finalcolumn>1). Down-regulated genes are shown in bold italics in Table 1(final column<1).

TABLE 1 Genes regulated by GW1516 treatment and exercise trainingFEATURE LOCUS DESCRIPTION GW + Tr ANGIOGENESIS 1417130_s_at Angptl4angiopoietin-like 4 5.495

CARBOHYDRATE METABOLISM 1449088_at Fbp2 fructose bisphosphatase 2 2.8081423439_at Pck1 phosphoenolpyruvate carboxykinase 1, cytosolic 3.5181434499_a_at Ldhb lactate dehydrogenase B 2.541 PROLIFERATION &APOPTOSIS 1425621_at Trim35 tripartite motif-containing 35 1.856

 

1448272_at Btg2 B-cell translocation gene 2, anti-proliferative 1.6011452260_at Cidec cell death-inducing DFFA-like effector c 4.7711417956_at Cidea cell death-inducing DNA fragmentation factor, alpha49.625  subunit-like effector A CYTOKINES 1426278_at Ifi27 interferon,alpha-inducible protein 27 1.714 1421239_at Il6st interleukin 6 signaltransducer 1.972 FAT METABOLISM 1448318_at Adfp adipose differentiationrelated protein 2.009 1424729_at BC054059 cDNA sequence BC054059 5.08 1424937_at 2310076L09Rik RIKEN cDNA 2310076L09 gene 1.868 1450010_atHsd17b12 hydroxysteroid (17-beta) dehydrogenase 12 2.376 1415965_at Scd1stearoyl-Coenzyme A desaturase 1 6.494 1415822_at Scd2 stearoyl-CoenzymeA desaturase 2 1.849 1423828_at Fasn fatty acid synthase 6.3231455061_a_at Acaa2 acetyl-Coenzyme A acyltransferase 2 (mitochondrial 3-1.926 oxoacyl-Coenzyme A thiolase) 1448987_at Acadl acetyl-Coenzyme Adehydrogenase, long-chain 2.549 1422651_at Adipoq adiponectin, C1Q andcollagen domain containing 3.082 1422820_at Lipe lipase, hormonesensitive 3.032 1449964_a_at Mlycd malonyl-CoA decarboxylase 1.7811426785_s_at Mgll monoglyceride lipase 1.907 1420658_at Ucp3 uncouplingprotein 3 (mitochondrial, proton carrier) 2.943 1425326_at Acly ATPcitrate lyase 2.606 1460409_at Cpt1a carnitine palmitoyltransferase 1a,liver 2.753 1422677_at Dgat2 diacylglycerol O-acyltransferase 2 2.784

1425834_a_at Gpam glycerol-3-phosphate acyltransferase, mitochondrial2.207 1417273_at Pdk4 pyruvate dehydrogenase kinase, isoenzyme 4 2.27 1449182_at Retn resistin 4.114 1435630_s_at Acat2 acetyl-Coenzyme Aacetyltransferase 2 1.625 1425829_a_at Abcb1a ATP-binding cassette,sub-family B (MDR/TAP), member 10.322  1A 1423166_at Cd36 CD36 antigen1.584 1422811_at Slc27a1 solute carrier family 27 (fatty acidtransporter), member 1 3.58  1416023_at Fabp3 fatty acid binding protein3, muscle and heart 1.833 1424155_at Fabp4 fatty acid binding protein 4,adipocyte 2.189 1431056_a_at Lpl lipoprotein lipase 1.659 1422432_at Dbidiazepam binding inhibitor 1.936 1422811_at Slc27a1 solute carrierfamily 27 (fatty acid transporter), 1 3.58  HEAT SHOCK RESPONSE1448881_at Hp haptoglobin 1.679 1427126_at Hspa1b heat shock protein 1B8.845 1438902_a_at Hsp90aa1 heat shock protein 90 kDa alpha (cytosolic),class A 1.513 member 1 1431274_a_at Hspa9a heat shock protein 9A 1.61 1416755_at Dnajb1 DnaJ (Hsp40) homolog, subfamily B, member 1 3.59 MISCELLANEOUS 1460256_at Car3 carbonic anhydrase 3 2.339 1415841_atDync1i2 dynein cytoplasmic 1 intermediate chain 2 1.705 1432344_a_atAplp2 amyloid beta (A4) precursor-like protein 2 1.937 1416429_a_at Catcatalase 1.82  1418306_at Crybb1 crystallin, beta B1 2.457 1448842_atCdo1 cysteine dioxygenase 1, cytosolic 3.266

1453527_a_at Neurl neuralized-like homolog (Drosophila) 1.941 1451603_atRtbdn retbindin 2.32  1453724_a_at Serpinf1 serine (or cysteine)peptidase inhibitor, clade F, member 1 7.765

1427285_s_at Surf4 surfeit gene 4 2.091 1424737_at Thrsp thyroid hormoneresponsive SPOT14 homolog (Rattus) 2.685 1431609_a_at Acp5 acidphosphatase 5, tartrate resistant 3.91  1448538_a_at D4Wsu53e DNAsegment, Chr 4, Wayne State University 53, 1.586 expressed

1425552_at Hip1r huntingtin interacting protein 1 related 1.75 

1429360_at Klf3 Kruppel-like factor 3 (basic) 1.901 1449413_at Mpv17lMpv17 transgene, kidney disease mutant-like 1.988 1451667_atC530043G21Rik RIKEN cDNA C530043G21 gene 1.5  1425865_a_at Lig3 ligaseIII, DNA, ATP-dependent 2.693 1415994_at Cyp2e1 cytochrome P450, family2, subfamily e, polypeptide 1 2.941 1417867_at Cfd complement factor D(adipsin) 2.828 1451015_at Tkt transketolase 2.256 1432344_a_at Aplp2amyloid beta (A4) precursor-like protein 2 1.937 1419487_at Mybph Myosinbinding protein H 1.578 MITOCHONDRIAL PROTEINS

1415897_a_at Mgst1 microsomal glutathione S-transferase 1 1.916

1423109_s_at Slc25a20 solute carrier family 25 (mitochondrial 1.865carnitine/acylcarnitine translocase), member 20

OXYGEN CARRIERS 1448348_at Gpiap1 GPI-anchored membrane protein 1 1.83 1451203_at Mb myoglobin 1.578

1428361_x_at Hba-a1 hemoglobin alpha, adult chain 1 1.632 1417184_s_atHbb-b2|Hbb-y hemoglobin, beta adult minor chain|hemoglobin Y, beta-1.626 like embryonic chain SIGNAL TRANSDUCTION

1455918_at Adrb3 adrenergic receptor, beta 3 3.83 

1452097_a_at Dusp7 dual specificity phosphatase 7 1.661 1419191_at Hipk3homeodomain interacting protein kinase 3 1.694 1448152_at Igf2insulin-like growth factor 2 1.635 1422313_a_at Igfbp5 insulin-likegrowth factor binding protein 5 1.772 1428265_at Ppp2r1b proteinphosphatase 2 (formerly 2A), regulatory subunit A 2.509 (PR 65), betaisoform

1449342_at Ptplb protein tyrosine phosphatase-like (proline instead of2.38  catalytic arginine), member b 1422119_at Rab5b RAB5B, member RASoncogene family 1.603

1425444_a_at Tgfbr2 transforming growth factor, beta receptor II 2.13 1431164_at Rragd Ras-related GTP binding D 2.101 1420816_at Ywhag3-monooxygenase/tryptophan 5-monooxygenase activation 1.87  protein,gamma polypeptide STEROID BIOGENESIS 1418601_at Aldh1a7 aldehydedehydrogenase family 1, subfamily A7 3.862 1426225_at Rbp4 retinolbinding protein 4, plasma 2.065

TRANSCRIPTION 1417794_at Zfp261 zinc finger protein 261 1.847 1424731_atNle1 notchless homolog 1 (Drosophila) 1.831 1454791_a_at Rbbp4retinoblastoma binding protein 4 2.865 1460281_at Asb15 ankyrin repeatand SOCS box-containing protein 15 1.78  1449363_at Atf3 activatingtranscription factor 3 1.802 1418982_at Cebpa CCAAT/enhancer bindingprotein (C/EBP), alpha 2.168 1417065_at Egr1 early growth response 12.577

1415899_at Junb Jun-B oncogene 1.792 1421554_at Lmx1a LIM homeoboxtranscription factor 1 alpha 4.106 1416959_at Nr1d2 nuclear receptorsubfamily 1, group D, member 2(Reverb-b) 1.794 1450749_a_at Nr4a2nuclear receptor subfamily 4, group A, member 2 (NURR1) 1.776 1460215_atRpo1-4 RNA polymerase 1-4 2.498

1420892_at Wnt7b wingless-related MMTV integration site 7B 4.4491423100_at Fos FBJ osteosarcoma oncogene 3.9  TRANSPORT PROTEINS

1425546_a_at Trf transferrin 1.907 1423743_at Arcn1 archain 1 1.6171451771_at Tpcn1 two pore channel 1 2.842 1416629_at Slc1a5 solutecarrier family 1 (neutral amino acid transporter), 1.939 member 51420295_x_at Clcn5 chloride channel 5 2.333 1417839_at Cldn5 claudin 51.545

1434617_x_at 1810073N04Rik RIKEN cDNA 1810073N04 gene 2.326 Data isaverage of N = 3 samples in each group (p < 0.05).

Surprisingly, the combination of GW1516 treatment and exerciseestablished a unique gene expression pattern that was neither anamalgamation nor a complete overlap of the two interventions (FIG. 6).This unique signature included 48 new target genes (Table 2) notregulated by GW1516 and exercise alone and excluded 74 genes regulatedby GW1516 or exercise alone (a selected few of which are shown in Table3). This signature for the combination of GW1516 treatment and exercise(Table 2) was highly enriched in genes encoding regulatory enzymes forenergy homeostasis, angiogenesis, oxygen transport, signal transduction,transcription and substrate transport, which are processes that areinvolved in endurance adaptation. Particularly, a predominance of genesinvolved in oxidative metabolism, is selectively up-regulated bycombined exercise and drug treatment (see unbolded genes in Tables 1 and2). In addition, several stress-related genes activated by eitherintervention, including heat shock proteins, metallothioneins and otherstress biomarkers (Table 3) are not changed by the combination possiblyreflecting a potential lessening of exercise-based damage.

TABLE 2 Gene targets unique to combined GW1516 treatment and exercisetraining. DESCRIPTION LOCUS GW + Tr ANGIOGENESIS

CARBOHYDRATE METABOLISM phosphoenolpyruvate carboxykinase 1, cytosolicPck1 3.518 CYTOKINES interferon, alpha-inducible protein 27 Ifi27 1.714FAT METABOLISM adipose differentiation related protein Adrp 2.009stearoyl-Coenzyme A desaturase 2 Scd2 1.849 acetyl-Coenzyme Aacetyltransferase 2 Acat2 1.625 ATP citrate lyase Acly 2.606adiponectin, C1Q and collagen domain containing Adipoq 3.082diacylglycerol O-acyltransferase 2 Dgat2 2.784

lipase, hormone sensitive Lipe 3.032 monoglyceride lipase Mgll 1.907resistin Retn 4.114 CD36 antigen Cd36 1.584 fatty acid binding protein4, adipocyte Fabp4 2.189 lipoprotein lipase Lpl 1.659 HEAT SHOCKRESPONSE haptoglobin Hp 1.679 MITOCHONDRIAL PROTEINS

microsomal glutathione S-transferase 1 Mgst1 1.916 OTHERS carbonicanhydrase 3 Car3 2.339 cysteine dioxygenase 1, cytosolic Cdo1 3.266 DNAsegment, Chr 4, Wayne State University 53, expressed D4Wsu53e 1.586dynein cytoplasmic 1 intermediate chain 2 Dync1i2 1.705

Kruppel-like factor 3 (basic) Klf3 1.901

thyroid hormone responsive SPOT14 homolog (Rattus) Thrsp 2.685cytochrome P450, family 2, subfamily e, polypeptide 1 Cyp2e1 2.941complement factor D (adipsin) Cfd 2.828 transketolase Tkt 2.256 OXYGENCARRIERS GPI-anchored membrane protein 1 Gpiap1 1.83

PROLIFERATION & APOPTOSIS

cell death-inducing DFFA-like effector c Cidec 4.771 SIGNAL TRANSDUCTION

dual specificity phosphatase 7 Dusp7 1.661 homeodomain interactingprotein kinase 3 Hipk3 1.694 insulin-like growth factor binding protein5 Igfbp5 1.772 protein phosphatase 2 (formerly 2A), regulatory subunit A(PR 65), beta Ppp2r1b 2.509 isoform protein tyrosine phosphatase-like(proline instead of catalytic arginine), Ptplb 2.38 member b STEROIDBIOGENESIS retinol binding protein 4, plasma Rbp4 2.065 TRANSCRIPTIONCCAAT/enhancer binding protein (C/EBP), alpha Cebpa 2.168 nuclearreceptor subfamily 1, group D, member 2(Reverb-b) Nr1d2 1.794 TRANSPORTtransferrin Trf 1.907 archain 1 Arcn1 1.617 solute carrier family 1(neutral amino acid transporter), member 5 Slc1a5 1.939 RIKEN cDNA1810073N04 gene 1810073N04Rik 2.326 Down-regulated genes are in bolditalics. (N = 3, each pooled from 3 mice, p < 0.05).

TABLE 3 Gene targets regulated by GW1516 treatment or exercise trainingalone. FEATURE LOCUS DESCRIPTION GW Tr GW + Tr Hspb1 heat shock protein1 1.815 1.965 — 1451284_at Hspb7 heat shock protein family, 7(cardiovascular) 3.414 1.753 — 1422943_a_at Dnaja1 DnaJ (Hsp40) homolog,subfamily A, 1 — 1.545 — 1421290_at Hsp110 heat shock protein 110 —1.587 — 1416288_at Serpinh1 serine (or cysteine) peptidase inhibitor, H,1 — 2.198 — 1423566_a_at Dnaja4 DnaJ (Hsp40) homolog, subfamily A, 41.756 1.545 — 1417872_at Mt1 metallothionein 1 2.364 — — 1424596_s_atMt2 metallothionein 2 2.151 — — 1416157_at Cryab crystallin, alpha B1.561 1.52  — 1423139_at Crygf crystallin, gamma F 1.801 3.56  —1448830_at Smad3 MAD homolog 3 (Drosophila) 1.841 1.886 — 1450637_a_atAnkrd1 ankyrin repeat domain 1 (cardiac muscle) 4.235 — — 1416029_atTnfrsf12a TNF receptor superfamily, 12a 1.759 1.782 — 1426464_at Jun Junoncogene — 1.521 — Data is average of N = 3 samples in each group (p <0.05)

Thirty-two percent of the GW+Tr-regulated genes encode enzymes ofmetabolic pathways such as fatty acid biosynthesis/storage (e.g., FAS,SCD 1 & 2), uptake [e.g., FAT/CD36, fatty acid binding proteins (FABP)and LPL] and oxidation [e.g., adiponectin, hormone sensitive lipase(HSL), PDK4, UCP3]; and carbohydrate metabolism [e.g., fructosebisphosphate 2 (FBP2), phosphoenolpyruvate carboxykinase 1 (PEPCK1),lactate dehydrogenase B], which along with oxygen transporters andmitochondrial proteins form the largest class of genes directly linkedto muscle performance (Ikeda et al., Biochem. Biophys. Res. Commun.296:395-400, 2002; Achten and Jeukendrup, Nutrition. 20:716-27, 2004;Hittel et al., J. Appl. Physiol. 98: 168-79, 2005; Civitarese et al.,Cell Metab. 4:75-87, 2006; Nadeau et al., FASEB J. 17:1812-9, 2006;Kiens, Physiol. Rev. 86:205-43, 2006; Yamauchi et al., Nat. Med.8:1288-95, 2006). Unexpectedly, established PPARα target genes fattyacyl-CoA oxidase and medium chain acyl-CoA dehydrogenase (MCAD) were notrepresented in the signature. All but four of these metabolic genes wereinduced, which indicated a general increase in oxidative capacity ofskeletal muscle in exercise-trained subjects that received GW1516treatment.

Other genes regulated in quadriceps muscle by the combination ofexercise and GW1516 treatment encoded proteins involved in pathways suchas angiogenesis (e.g., angiopoietin-like 4 protein/also a knownregulator of lipid metabolism), (e.g., adrenergic receptor β3,insulin-like growth factor, insulin-like growth factor binding protein5), transcription (e.g., C/EBP α, Reverb β, NURR1) and substratetransport (e.g., transferrin, chloride channel 5) (Nagase et al., J.Clin. Invest. 97:2898-904, 1996; Singleton and Feldman, Neurobiol. Dis.8:541-54, 2001; Adams, J. Appl. Physiol. 93:1159-67, 2002; Centrella etal., Gene. 342: 13-24, 2004; Lundby et al., Eur. J. Appl. Physiol. 96:363-9, 2005; Mahoney et al., FASEB J. 19:1498-500, 2005; Mahoney et al.,Phys. Med. Rehabil. Clin. N. Am. 16: 859-73, 2005; Ramakrishnan et al.,J. Biol. Chem. 280:8651-9, 2005). Without wishing to be bound to aparticular theory, such other genes are likely involved, at least inpart, in muscle remodeling and increased endurance observed inGW1516-treated, exercise-trained subjects.

Interestingly, comparative expression analysis of the 48 gene subset ofthe endurance signature (Table 2), but not of either intervention alone,revealed a striking similarity to ‘untrained’ VP16-PPARδ transgenicmice. This observation confirms the primary dependence of the 48 geneson PPARδ and indicates that exercise-generated signals may function tosynergize PPARδ transcriptional activity to levels comparable totransgenic over-expression. Therefore, exercise cues along with PPARδagonist may function to hyper-activate receptor transcriptional activityto re-program of adult muscle.

Genes and/or proteins uniquely affected (e.g., up-regulated ordown-regulated or not substantially regulated) by exercise in thepresence of one or more pharmaceutical agents (e.g., PPARδ agonists) canbe used as markers, for instance, of “drug doping” in exercise-trainedsubjects (e.g., athletes). It is expected that the unique set of 48genes regulated by GW+Tr, but not GW1516 treatment or exercise trainingalone, can be used to identify exercised subjects who have received avariety performance-enhancing drugs.

Example 6 PPARδ Directly Interacts with Exercise-Activated Kinases,p44/42 MAPK and AMPK

Exercise training is known to activate kinases, such as p44/42 MAPK andAMPK, which regulate gene expression in skeletal muscle (Chen et al.,Diabetes, 52:2205-12, 2003; Goodyear et al., Am. J. Physiol.,271:E403-8, 1996). AMPK affects skeletal muscle gene expression andoxidative metabolism (Chen et al., Diabetes. 52: 2205-12, 2003, Reznicket al., J. Physiol. 574: 33-9, 2006). The interaction betweenexercise-regulated kinases and PPARδ signaling is described in thisExample.

The levels of phospho-p44/42 MAPK and phospho-AMPK α subunit and totalAMPK were determined in protein homogenates of quadriceps muscle byWestern blot. Antibodies specific for phospho-p44/42 MAPK, phospho- andtotal-AMPK α1 antibodies were obtained from Cell Signaling. Thephospho-specific AMPK α1 antibody recognizes the key activatingthreonine in the activation loop.

Active forms of both kinases (phospho-p44/42 MAPK and phospho-AMPK αsubunit) were expressed at higher levels in the quadriceps muscles ofexercised mice relative to the sedentary controls (FIG. 7A). Previousreports claim that PPARδ is not required for activation of AMPK byGW1516 in cultured cells (Kramer et al, Diabetes. 54(4):1157-63, 2005and Kramer et al., J. Biol. Chem. 282(27):19313-2, 2007). In contrast,it was observed that GW1516 failed to activate p44/42 or AMPK in eithersedentary or trained muscles, which indicated that PPARδ-regulatedeffects are downstream to the exercise-induced signals that activatethese kinases. Furthermore, AMPK appears to be constitutively active inmuscles of VP16-PPARδ transgenic mice in absence of exercise or drug(FIG. 7B). These results indicate that synergy is AMPK and PPARδco-dependent.

If synergy is AMPK and PPARδ co-dependent, selective co-activation ofAMPK and PPARδ would induce gene expression changes that mimic thosetriggered by combined exercise and PPARδ as well as VP16-PPARδover-expression. To demonstrate this, transcriptional changes induced inskeletal muscle by combined exercise and GW1516 treatment (as describedin Example 5) were compared to that of combined AMPK activator (the cellpermeable AMP analog AICAR; 250 mg/kg/day, i.p.) and GW1516 (5mg/kg/day, oral gavage) treatment for 6 days. Genome analysis wasperformed using the methods described in Example 5.

Simultaneous GW1516 and AICAR treatment for 6 days created a unique geneexpression signature in the quadriceps of untrained C57B1/6J mice (FIG.8A, which includes target genes associated with translation, proteinprocessing, amino acid metabolism, fat metabolism, oxygen carriers,carbohydrate metabolism, signal transduction, transcription, transport,steroid biogenesis, heat shock response, angiogenesis, proliferation andapoptosis, cytokines, contractile proteins, stress, and others) thatshares 40% of the genes with that of combined GW1516 treatment andexercise (FIG. 8B). Classification of the 52 genes common to the twosignatures (combined PPARδ activation and exercise or PPARδ and AMPKco-activation) (listed in Table 4) revealed that the majority of thetargets were linked to oxidative metabolism.

TABLE 4 Targets common to exercise-PPARδ and AMPK-PPARδ gene signatures.DESCRIPTION LOCUS Tr + GW AI + GW ANGIOGENESIS angiopoietin-like 4Angptl4 5.495 2.917 APOPTOSIS cell death-inducing DFFA-like effector cCidec 4.771 1.838 cell death-inducing DNA fragmentation factor, alphaCidea 49.625 1.842 subunit-like effector A CARBOHYDRATE METABOLISMlactate dehydrogenase B Ldhb 2.541 1.917 fructose bisphosphatase 2 Fbp22.808 2.478 FAT METABOLISM stearoyl-Coenzyme A desaturase 1 Scd1 6.4941.78 fatty acid binding protein 3, muscle and heart Fabp3 1.833 1.5pyruvate dehydrogenase kinase, isoenzyme 4 Pdk4 2.27 2.486 uncouplingprotein 3 (mitochondrial, proton carrier) Ucp3 2.943 2.792 adiponectin,C1Q and collagen domain containin Adipoq 3.082 1.56 diacylglycerolO-acyltransferase 2 Dgat2 2.784 2.14 solute carrier family 27 (fattyacid transporter), member 1 Slc27a1 3.58 2.195 lipase, hormone sensitiveLipe 3.032 1.746 solute carrier family 25 (mitochondrial Slc25a20 1.7041.697 carnitine/acylcarnitine translocase), member 20 CD36 antigen Cd361.584 1.513 phosphoenolpyruvate carboxykinase 1, cytosolic Pck1 3.5181.781 fatty acid synthase Fasn 6.323 2.24 fatty acid binding protein 4,adipocyte Fabp4 2.189 1.81 monoglyceride lipase Mgll 1.907 1.51acetyl-Coenzyme A acetyltransferase 2 Acat2 1.625 1.563 acetyl-CoenzymeA dehydrogenase, long-chain Acadl 2.549 1.992 resistin Retn 4.114 1.756malonyl-CoA decarboxylase Mlycd 1.781 1.962 transketolase Tkt 2.2561.983 ATP citrate lyase Acly 2.458 1.91 HEAT SHOCK heat shock protein 90kDa alpha (cytosolic), class A member 1 Hsp90aa1 1.455 0.616 DnaJ(Hsp40) homolog, subfamily B, member 1 Dnajb1 3.59 0.604 CYTOKINESinterferon, alpha-inducible protein 27 Ifi27 1.714 1.537 OTHERsarcolipin Sln 0.363 4.576 thyroid hormone responsive SPOT14 homolog(Rattus) Thrsp 2.685 1.766 RIKEN cDNA 2310076L09 gene 2310076L09Rik1.868 2.117 myosin, heavy polypeptide 2, skeletal muscle, adult Myh22.194 1.797 surfeit gene 4 Surf4 2.091 0.654 acid phosphatase 5,tartrate resistant Acp5 3.91 1.477 serine (or cysteine) proteinaseinhibitor, clade A, member 1a Serpina1a 0.396 3.891 cysteine dioxygenase1, cytosolic Cdo1 3.266 1.678 erythroid differentiation regulator 10.619 1.805 RIKEN cDNA 1810073N04 gene 1810073N04Rik 2.326 1.628superoxide dismutase 3, extracellular Sod3 1.606 1.617 complement factorD (adipsin) Cfd 2.828 1.5 cytochrome P450, family 2, subfamily e,polypeptide 1 Cyp2e1 2.941 1.743 catalase Cat 1.728 1.902 early growthresponse 1 Egr1 2.577 0.65 OXYGEN CARRIER hemoglobin, beta adult minorchain|hemoglobin Y, beta- Hbb-b2|Hbb-y 1.626 1.503 like embryonic chainSTEROID BIOGENESIS retinol binding protein 4, plasma Rbp4 2.065 2.225SIGNAL TRANSDUCTION adreneyrgic receptor, beta 3 Adrb3 3.83 1.56 proteintyrosine phosphatase-like (proline instead of catalytic Ptplb 2.38 1.569arginine), member b dual specificity phosphatase 7 Dusp7 1.661 1.672TRANSCRIPTION nuclear receptor subfamily 4, group A, member 2 Nr4a21.776 0.437 TRANSPORT solute carrier family 1 (neutral amino acidtransporter), Slc1a5 1.939 1.511 member 5 two pore channel 1 Tpcn1 2.8421.487 seminal vesicle secretion 5 Svs5 0.095 2.243 Data is average of N= 3 samples in each group (p < 0.05).

Quantitative expression analysis of selective oxidative genes (eight ofthose listed in Table 4) was determined in quadriceps of mice treatedwith vehicle (V), GW1516 (GW, 5 mg/kg/day), AICAR (AI, 250 mg/kg/day)and the combination of the two drugs (GW+AI) for 6 days using themethods described in Example 1. As shown in FIGS. 9A-H, several of thesebiomarkers including PDK4, SCD1, ATP citrate lyase, HSL, mFABP and LPLwere induced in a synergistic fashion by GW1516 and AICAR in thequadriceps (FIGS. 9C-9H). Intriguingly, synergism was undetectable inUCP3 and mCPT I (FIGS. 9A and B). These genes were induced in quadricepsof untrained VP16-PPARδ mice, where AMPK is constitutively active (Table5).

TABLE 5 Selective oxidative genes induced in muscle by combined PPARδand AMPK activation as well as VP16-PPARδ over-expression DescriptionLocus GW + AI VP-PPARδ ATP citrate lyase Acly 1.648 3.095 carnitinepalmitoyltransferase 1b, Cpt1b 1.371 1.678 muscle fatty acid bindingprotein 3, muscle Fabp3 1.447 5.904 and heart fatty acid synthase Fasn2.24 2.749 lipoprotein lipase Lpl 1.113 1.72 lipase, hormone sensitiveLipe 1.746 2.203 pyruvate dehydrogenase kinase, Pdk4 2.486 5.06isoenzyme 4 stearoyl-Coenzyme A desaturase 1 Scd1 1.78 7.353 uncouplingprotein 3 Ucp3 2.792 4.107

Collectively, these results demonstrate that while interaction betweenAMPK and PPARδ may substantially contribute to re-programming of theskeletal muscle transcriptome during exercise, additional changes mayinvolve cross-talk between other components of the exercise signalingnetwork and PPARδ.

In summary, PPARδ and exercise synergistically regulate runningendurance. Although not bound by theory, kinase activation may influencePPARδ signaling during exercise in establishing an “endurance geneexpression signature” that effectively enhances performance.

Example 7 AMPK Increases Transcriptional Activation by PPARδ

The genetic synergism described in Example 6 indicates that AMPKdirectly regulates the transcriptional activity of PPARδ in skeletalmuscles. To demonstrate this, an analysis of the effects of GW1516 andAICAR on gene expression in primary muscle cells isolated from wild typeand PPARδ null mice was performed.

Primary muscle cells were isolated from wild type and PPARδ null mice aspreviously described (Rando and Blau, J. Cell. Biol. 125(6):1275-87,1994). Skeletal muscle C2C12 cells were cultured in DMEM containing 20%serum and penicillin/streptomycin cocktail. For differentiation, cellsat 80% confluence were switched to a differentiation medium (DMEM+2%serum) for 4 days to obtain differentiated myotubules. Cells weretreated with vehicle, GW1516, AICAR, or GW1516+AICAR (GW: 0.1 μM; AICAR:500 μM) for 24 hours. RNA expression of UCP3, PDK4, LPL, and HSL wasdetermined using real time quantitative PCR as described in Example 1.

As shown in FIGS. 10A-D, synergism is dependent on PPARδ and lost in thenull cells. Similar synergistic regulation of gene expression by GW1516and AICAR was also observed in differentiated C2C12 cells. These resultsshow that AMPK activation may enhance ligand-dependent transcriptionaleffects of PPARδ in muscles.

To more directly address this, reporter gene expression assays wereutilized. AD 293 cells were cultured in DMEM containing 10% serum and anantibiotic cocktail. Cells were transfected with one or more ofCMX-Flag, CMX-Flag PPARδ, CMX-Tk-PPRE, or CMX-βGAL, or an hAMPK (α1 andα2 subunits, Origene) expression vector using Lipofectamine™ 2000 inaccordance with the manufacturer's instructions. Anti-Flagantibody-conjugated beads were incubated overnight at 4° C. with lysatesfrom transfected cells. Flag-tagged protein or protein complexes wereimmunoprecipitated by separating the beads from non-bound materials. Thebeads were washed in ice-cold lysis buffer followed by extraction inLaemmli buffer. For co-immunoprecipitation experiments SDS was excludedfrom the lysis buffer. Western blotting was performed with antibodiesspecific for the Flag tag or AMPK α subunit(s).

Co-transfection of either catalytic AMPK α1 or α2 subunits, but notcontrol vector, with PPARδ increased the basal (FIG. 10E) andGW1516-dependent transcriptional activity (FIG. 10F) of PPARδ ininducing a PPRE-driven reporter gene in AD293 cells. AMPKover-expression or GW1516 treatment did not change reporter activity intransfections excluding the PPARδ expression vector negating thepossibility of an effect via RXR. Additional results indicate that AMPKmay modulate PPARδ transcriptional activity by directly interacting withthe receptor. In AD293 cells co-transfected with Flag-PPARδ and witheither catalytic AMPK α1 or α2 subunits, both of the subunitsco-immunoprecipitated as a complex with Flag-PPARδ (FIG. 10G).Furthermore, Flag-PPARδ also co-immunoprecipitated endogenous AMPKαsubunits from AD293 cells confirming a direct physical interactionbetween the nuclear receptor and the kinase (FIG. 10H). Despite physicalinteraction, AMPK failed to increase PPARδ phosphorylation.

While potential AMPK phosphorylation sites were found in PPARδ, none ofthese sites were phosphorylated by AMPK in in vitro kinase assays. Thiswas further confirmed by measuring the p32 labeling of PPARδ in AD 293cells in the presence or absence of AMPK. AD 293 cells were transfectedwith PPARδ and hAMPk (α1 or α2 subunit) expression vectors as describedabove. Forty-eight hours after transfection, the cells were washed threetimes with phosphate-free DMEM and incubated with ³²P-orthophosphate inphosphate-free DMEM for 20 hours (100 μCi/5 ml). Cells were washed threetimes with ice-cold phosphate-free DMEM and lysed in ice-cold lysisbuffer.

As shown in FIG. 10I, overall PPARδ phosphorylation is not increased byAMPK in vivo. However, co-transfection of AMPKα2 and co-activator PGC1α(a known phosphorylation target of AMPK) co-operatively interact tofurther induce both the basal and ligand-dependent transcriptionalactivity of PPARδ (FIG. 10J). Strikingly, no significant physicalinteraction between Flag-PGC1α and AMPK (FIG. 10K) was detected, both ofwhich independently interacted with PPARδ. Collectively, theseobservations indicate that AMPK may be present in a transcriptionalcomplex with PPARδ where it can potentiate receptor activity via directprotein-protein interaction and/or by phosphorylating co-activators suchas PGC1α.

These results indicate that AMPK directly interacts with PPARδ anddramatically increases basal and ligand-dependent transcription via thereceptor. Despite physical interaction, AMPK does not phosphorylatePPARδ. AMPK and its substrate PGC1α synergistically increased PPARδtranscription, indicating indirect regulation of receptor by AMPK viaco-regulator modification.

The conclusion that exercise-activated AMPK interacts with PPARδ inregulating gene expression in vivo is strengthened by the observationthat treatment of animals with AICAR (AMPK activator) and GW1516 createsa gene signature in skeletal muscle that replicates up to 40% of thegenetic effects of combined exercise and GW1516 treatment (see Table 4).Moreover, several candidate genes from this signature aresynergistically induced by GW1516 and AICAR in wild type but not inPPARδ null primary muscle cells, demonstrating that the interactiveeffects of the two drugs are mediated through PPARδ. While 45% of thecommonly regulated genes are linked to oxidative metabolism, additionalcommon targets relevant to muscle performance include angiogenic, signaltransduction and glucose sparing genes (Table 4). It is possible thatthe portion of the PPARδ-exercise signature that is independent ofPPARδ-AMPK interaction (FIG. 8B) may depend on cross-talk between thereceptor and other exercise signal transducers such as MAPK,calcineurin/NFAT and SIRT 1. These possibilities are summarized in FIG.10L, where AMPK and additional components of the signaling network areproposed to interact with liganded PPARδ to generate a muscle endurancegene signature and enhanced endurance adaptation.

In summary, it is shown herein that synthetic PPARδ activation aloneinduces a set of genomic changes that fail to alter the preset musclearchitecture and endurance levels in adult mice. However, thecombination of PPARδ activation with exercise brings about noveltranscriptional changes, potentially via interaction with kinases suchas AMPK (as depicted in FIG. 10L), re-setting the muscle transcriptometo a phenotype that dramatically enhances muscle performance.

Example 8 Enhancing Exercise Effect in a Subject

This example describes methods that can be used to increase or enhancean exercise in a healthy mammalian subject. Although specific conditionsare described, one skilled in the art will appreciate that minor changescan be made to such conditions.

Healthy adult human subjects perform aerobic exercise (e.g., running)for at least 30 minutes (e.g., 30-90 minutes) for at least 3-4 days perweek (e.g., 3-7 days per week) for at least 2 weeks (e.g., at least 4-12weeks). The exercise is performed at 40%-50% maximal heart rate, 50%-60%maximal heart rate, 60%-70% maximal heart rate, or 75%-80% maximal heartrate, where maximum heart rate for a human subject is calculated as: 220bps—(age of the subject).

During or after performing aerobic exercise as described above, thesubjects are orally administered GW1516[(2-methyl-4(((4-methyl-2-(4-trifluoromethylphenyl)-1,3-thiazol-5-yl)methyl)sulfanyl)phenoxy)aceticacid] at a dose of 1 to 20 mg per day, such as 2.5 or 10 mg per day.Subjects can continue to perform aerobic exercise while receivingGW1516. The subject can receive GW1516 for a period of at least 2 weeks,such as at least 4 weeks.

The exercise effect achieved in the treated subjects (e.g., runningendurance) can be compared to such an effect in untreated subjects.Exercise effect can be measured using methods known in the art, such asmeasuring aerobic or running endurance (for example measuring distancerun until exhaustion or amount of time to run a particular distance). Insome instances, the exercise effect of interest is increased in treatedsubjects by at least 5%, such as at least 10% as compared to untreatedsubjects.

Example 9 Identifying Performance Enhancing Substances in anExercise-Trained Subject

This example describes methods that can be used to identifyperformance-enhancing substances in an exercised-trained subject.

A biological sample obtained from a healthy adult human is analyzed todetermine if the subject is taking a PES (e.g., GW1516) by analyzingexpression of one or more of the molecules (nucleic acids or proteins)listed in Table 2 or Table 4. Suitable biological samples includesamples containing genomic DNA or RNA (including mRNA) or proteinsobtained from cells of a subject, such as those present in peripheralblood, urine, saliva, tissue biopsy, or buccal swab. For example, abiological sample of the subject can be assayed for a change inexpression (such as an increase or decrease) of any combination of atleast four molecules (nucleic acids or proteins) listed in Table 2 or 4,such as any combination of at least 10, at least 20, at least 30, or atleast 40 of those listed in Table 2 or 4, for example all of thoselisted in Table 2 or 4.

Analyzing Nucleic Acid Molecules

Methods of isolating nucleic acid molecules from a biological sample areroutine, for example using PCR to amplify the molecules from the sample,or by using a commercially available kit to isolate mRNA or cDNA.However, nucleic acids need not be isolated prior to analysis. Nucleicacids can be contacted with an oligonucleotide probe that will hybridizeunder stringent conditions with one or more nucleic acid molecule listedin Table 2 or 4. The nucleic acids which hybridize with the probe arethen detected and quantified. The sequence of the oligonucleotide probecan bind specifically to a nucleic acid molecule represented by thesequences listed in Table 2 or 4.

Increased or decreased expression of the molecules listed in Table 2 or4 can be detected by measuring the cellular levels of mRNA. mRNA can bemeasured using techniques well known in the art, including for instanceNorthern analysis, RT-PCR and mRNA in situ hybridization. Details ofmRNA analysis procedures can be found, for instance, in providedexamples and in Sambrook et al. (ed.), Molecular Cloning: A LaboratoryManual, 2nd ed., vol. 1-3, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989.

Oligonucleotides specific to sequences listed in Table 2 or 4 can bechemically synthesized using commercially available machines. Theseoligonucleotides can then be labeled, for example with radioactiveisotopes (such as ³²P) or with non-radioactive labels such as biotin(Ward and Langer et al., Proc. Natl. Acad. Sci. USA 78:6633-57, 1981) ora fluorophore, and hybridized to individual DNA samples immobilized onmembranes or other solid supports by dot-blot or transfer from gelsafter electrophoresis. These specific sequences are visualized, forexample by methods such as autoradiography or fluorometric (Landegren etal., Science 242:229-37, 1989) or colorimetric reactions (Gebeyehu etal., Nucleic Acids Res. 15:4513-34, 1987).

Analyzing Proteins

Proteins in the biological sample can also be analyzed. In someexamples, proteins are isolated using routine methods prior to analysis.

In one example, surface-enhanced laser desorption-ionizationtime-of-flight (SELDI-TOF) mass spectrometry is used to detect changesin differential protein expression, for example by using theProteinChip™ (Ciphergen Biosystems, Palo Alto, Calif.). Such methods arewell known in the art (for example see U.S. Pat. No. 5,719,060; U.S.Pat. No. 6,897,072; and U.S. Pat. No. 6,881,586). SELDI is a solid phasemethod for desorption in which the analyte is presented to the energystream on a surface that enhances analyte capture or desorption.Therefore, in a particular example, the chromatographic surface includesantibodies that recognize proteins listed in Table 2 or 4. Antigenspresent in the sample can recognize the antibodies on thechromatographic surface. The unbound proteins and mass spectrometricinterfering compounds are washed away and the proteins that are retainedon the chromatographic surface are analyzed and detected by SELDI-TOF.The MS profile from the sample can be then compared using differentialprotein expression mapping, whereby relative expression levels ofproteins at specific molecular weights are compared by a variety ofstatistical techniques and bioinformatic software systems.

In another examples, the availability of antibodies specific to themolecules listed in Table 2 or 4 facilitates the detection andquantification of proteins by one of a number of immunoassay methodsthat are well known in the art, such as those presented in Harlow andLane (Antibodies, A Laboratory Manual, CSHL, New York, 1988). Methods ofconstructing such antibodies are known in the art. Any standardimmunoassay format (such as ELISA, Western blot, or RIA assay) can beused to measure protein levels. Immunohistochemical techniques can alsobe utilized for protein detection and quantification. For example, atissue sample can be obtained from a subject, and a section stained forthe presence of the desired protein using the appropriate specificbinding agents and any standard detection system (such as one thatincludes a secondary antibody conjugated to horseradish peroxidase).General guidance regarding such techniques can be found in Bancroft andStevens (Theory and Practice of Histological Techniques, ChurchillLivingstone, 1982) and Ausubel et al. (Current Protocols in MolecularBiology, John Wiley & Sons, New York, 1998).

For the purposes of detecting or even quantifying protein or nucleicacid expression, expression in the test sample can be compared to levelsfound in cells from a subject who has not taken a PES. Alternatively,the pattern of expression identified in the test subject can be comparedto that shown in Table 2 or 4.

For example, if the test sample shows a pattern of expression similar tothat in Table 2 or 4 (e.g., the genes shown as upregulated anddownregulated in Table 2 or 4 are observed in the subject to beupregulated and downregulated, respectively), this indicates that thesubject is taking a PES, such as a PPARδ agonist (e.g., GW1516). Incontrast, If the pattern of expression identified in the test subject isdifferent to that shown in Table 2 or 4 (e.g., the genes shown asupregulated and downregulated in Table 2 or 4 are observed in thesubject to be not differentially expressed or show a different patternof regulation), this indicates that the subject is not taking a PES,such as a PPARδ agonist (e.g., GW1516).

A significant increase in the non-bolded proteins listed in Table 2 inthe cells of a test subject compared to the amount of the same proteinfound in normal human cells is usually at least 2-fold, at least 3-fold,at least 4-fold or greater difference. Substantial overexpression of thenon-bolded proteins listed in Table 2 in the subject's sample can beindicative of the subject taking a PES. Similarly, a significantdecrease in the bolded proteins listed in Table 2 in the cells of a testsubject compared to the amount of the same protein found in normal humancells is usually at least 2-fold, at least 3-fold, at least 4-fold orgreater difference. Substantial underexpression of the bolded proteinslisted in Table 2 in the subject's sample can be indicative of thesubject taking a PES.

While this disclosure has been described with an emphasis uponparticular embodiments, it will be obvious to those of ordinary skill inthe art that variations of the particular embodiments may be used and itis intended that the disclosure may be practiced otherwise than asspecifically described herein. Accordingly, this disclosure includes allmodifications encompassed within the spirit and scope of the disclosureas defined by the following claims:

We claim:
 1. A method for enhancing an exercise effect in a subject,comprising performing by a subject physical activity sufficient toproduce an exercise effect; and administering to the subject aneffective amount of a PPARδ agonist, thereby enhancing the exerciseeffect in the subject.
 2. The method of claim 1, wherein the subject isa mammal.
 3. The method of claim 2, wherein the subject is a racingmammal.
 4. The method of claim 3, wherein the racing mammal is a horse,a dog, or a human.
 5. The method of claim 1, wherein the subject is anadult.
 6. The method of claim 1, wherein the subject is anexercise-trained subject.
 7. The method of claim 1, wherein the PPARδagonist is GW1516.
 8. The method of claim 1, wherein the PPARδ agonistis administered on the same day(s) on which the physical activity isperformed.
 9. The method of claim 1, wherein the physical activity is anaerobic exercise.
 10. The method of claim 9, wherein the aerobicexercise is running.
 11. The method of claim 9, wherein the exerciseeffect is improved running endurance.
 12. The method of claim 11,wherein improved running endurance is improved running distance orimproved running time or a combination thereof.
 13. The method of claim1, wherein the effective amount is from about 5 mg/kg per day to about10 mg/kg per day in a single dose or in divided doses.
 14. The method ofclaim 1, wherein administration comprises oral administration,intravenous injection, intramuscular injection, or subcutaneousinjection.
 15. The method of claim 1, wherein the exercise effect isincreased fatty acid oxidation in at least one skeletal muscle of thesubject.
 16. The method of claim 1, wherein the exercise effect is bodyfat reduction.
 17. The method of claim 16, wherein the body fat is whiteadipose tissue.
 18. A method for identifying the use ofperformance-enhancing substances in an exercise-trained subjectcomprising determining in a biological sample taken from anexercise-trained subject the expression of one or more molecules listedin Tables 2 or
 4. 19. The method of claim 18, wherein: (i) expression isupregulated in one or more of adipose differentiation related protein;stearoyl-Coenzyme A desaturase 2; acetyl-Coenzyme A acetyltransferase 2;ATP citrate lyase; adiponectin, C1Q and collagen domain containing;diacylglycerol O-acyltransferase 2; lipase, hormone sensitive;monoglyceride lipase; resistin; CD36 antigen; fatty acid binding protein4, adipocyte; lipoprotein lipase; microsomal glutathione S-transferase1; GPI-anchored membrane protein 1; dual specificity phosphatase 7;homeodomain interacting protein kinase 3; insulin-like growth factorbinding protein 5; protein phosphatase 2 (formerly 2A), regulatorysubunit A (PR 65), beta isoform; protein tyrosine phosphatase-like(proline instead of catalytic arginine); member b; CCAAT/enhancerbinding protein (C/EBP), alpha; nuclear receptor subfamily 1, group D,member 2(Reverb-b); transferring; archain 1; solute carrier family 1(neutral amino acid transporter), member 5; RIKEN cDNA 1810073N04 gene;haptoglobin; retinol binding protein 4, plasma; phosphoenolpyruvatecarboxykinase 1, cytosolic; cell death-inducing DFFA-like effector c;interferon, alpha-inducible protein 27; carbonic anhydrase 3; cysteinedioxygenase 1, cytosolic; DNA segment, Chr 4, Wayne State University 53,expressed; dynein cytoplasmic 1 intermediate chain 2; Kruppel-likefactor 3 (basic); thyroid hormone responsive SPOT14 homolog (Rattus);cytochrome P450, family 2, subfamily e, polypeptide 1; complement factorD (adipsin); and/or transketolase; or (ii) expression is downregulatedin one or more of gamma-glutamyl carboxylase; 3-oxoacid CoA transferase1; solute carrier family 38, member 4; annexin A7; CD55 antigen, RIKENcDNA 1190002H23 gene; fusion, derived from t(12; 16) malignantliposarcoma (human); lysosomal membrane glycoprotein 2; and/or neighborof Punc E11; or (iii) a combination of (i) and (ii).
 20. The method ofclaim 18, wherein determining expression comprises determining proteinexpression, determining expression of a gene encoding the protein, or acombination thereof.
 21. The method of claim 20, comprising determiningexpression of a gene encoding the protein.
 22. The method of claim 18,wherein the biological sample is a skeletal muscle biopsy.
 23. A methodof identifying an agent having potential to enhance exercise performancein a subject, comprising: providing a first component comprising a PPARδreceptor or an AMPK-binding fragment thereof; providing a secondcomponent comprising an AMP-activated protein kinase (AMPK), AMPKα1,AMPKα2, or a PPARδ-binding fragment of any thereof; contacting the firstcomponent and the second component with at least one test agent underconditions that would permit the first component and the secondcomponent to specifically bind to each other in the absence of the atleast one test agent; and determining whether the at least one testagent affects specific binding of the first component and the secondcomponent to each other, wherein an effect on specific bindingidentifies the at least one test agent as an agent having potential toenhance exercise performance in a subject.
 24. The method of claim 23,further comprising providing a third component comprising a PPARδagonist; and contacting the first component, second component, and thirdcomponent.
 25. The method of claim 24, wherein the PPARδ agonist isGW1516.